Cell Surface Vimentin Research Articles

203 IDENTIFICATION OF A POTENTIAL MULTILINEAGE-DIFFERENTIATING STRESS ENDURING CELL POPULATION IN PORCINE FETAL FIBROBLASTS

Physiologically, humans and pigs are very similar, which makes cell model systems such as porcine-induced pluripotent stem (iPS) cells an attractive model for studying human diseases. Recently, it has been shown that a finite population of cells, termed multilineage-differentiating stress enduring (MUSE) cells, which are particularly prone for reprogramming, exists in human fibroblasts. Human MUSE cells co-express the cell surface markers, SSEA-3 and CD105; however, it is unknown if a similar subpopulation of cells exists in porcine fibroblasts. Therefore, we examined the expression level of varying cell surface markers, including SSEA-1, SSEA-3, CD105, and Vimentin (positive control), in 9 porcine fetal fibroblast lines from 3 different breed backgrounds (Danish Landrace lines 2, 4, 5; Gottingen minipig lines A1, B2, B3; Yucatan minipig lines 1, 3, 4) by performing chromogen immunocytochemistry. The results revealed that all the 9 cell lines were 100% positive for Vimentin and negative for SSEA-3. However, a small positive population of SSEA-1 cells could be detected in Danish Landrace lines 2, 4, 5 (0.267%, 0.441%, 0.292%, respectively) and in Gottingen minipig lines A1, B2, B3 (0.207%, 0.214%, 0.325%, respectively). Furthermore, CD105 could only be detected in the Yucatan minipig lines (0.170, 0.177, and 0.233%, in lines 1, 3, and 4, respectively). To identify if any of these lines were more receptive to reprogramming, we performed non-integrating vector-based reprogramming. A total of 1 × 105 cells from each cell line was electroporated with 3 episomal plasmids (pCXLE-hOCT3/4-shp53; pCXLE-hSK; pCXLE-hUL), which were diluted to 1 µg µL–1, and then plated in DMEM-AQ + 10% FBS without antibiotics. The medium was replaced with the addition of penicillin/streptomycin on Day 2, and on Day 7 cells were trypsinized and passaged onto mitomycin C-treated mouse embryonic fibroblasts in iPS culture medium (DMEM-F12 + 20%KSR + NEAA + Pen/strep + 2 µL mL–1 β-Me + 10 ng mL–1 bFGF). Only a select number of Danish Landrace lines 2 and 4 formed ES-like colonies after 14 days, and few colonies with neural-like morphology emerged in Yucatan minipig line 3, but all these colonies lost their proliferative potential after a few passages. The reprogramming efficiency observed on Day 21 was 0.0787, 0.0170, and 0.0033% for Danish Landrace 2, Danish Landrace 4, and Yucatan 3 lines, respectively. The NANOG immunostaining was also performed at passage 1, and a minor proportion of the colonies in Danish Landrace line 2 was found to contain small populations of cells with nuclear staining for NANOG. Interestingly, Danish Landrace lines 2 and 4, which formed ES-like colonies, also expressed SSEA1, which may suggest that SSEA1 could potentially be a marker of a MUSE cell population. Further studies are ongoing to investigate this hypothesis. This research may further support the elite hypothesis for how reprogramming may occur.

Endothelial cell surface vimentin binding peptide induces angiogenesis under hypoxic/ischemic conditions

We have previously identified several angiogenic peptides that bind cell surface proteins by screening a phage display peptide library on human umbilical endothelial cells exposed to hypoxic conditions. In this study we describe one of the selected peptides, SP. We found by protein precipitation of endothelial cell lysates that the 12 amino acid SP peptide binds cell surface vimentin. Surprisingly, vimentin was detected on the cell surface of about 30% of intact endothelial cells under both normoxic and hypoxic conditions, as was demonstrated by fluorocytometric analysis on viable cells. The assessment of SP in the induction of angiogenesis was established by a significant increase in endothelial cell proliferation and tube formation under hypoxic conditions and not under normoxic conditions. Cell proliferation and tube length increased two-fold in endothelial cells in the presence of 10ng/ml SP peptide when compared to controls. The specificity of SP binding to vimentin was demonstrated by SP inhibition of anti-vimentin binding and by the inhibition of tube formation in cells transfected with siRNA against vimentin. Local intramuscular administrations of the peptide SP to ischemic hind limbs using the mouse hind limb ischemia model, demonstrated that SP inoculated at 1 and 10μg, improved blood perfusion compared to inoculations with an irrelevant peptide or PBS. The recovery of blood perfusion correlated with the increase in the number of detectable capillaries in the ischemic limb. The development of novel peptides for the induction of pro-angiogenic activity may pave the way for new therapeutic strategies in the treatment of cardiovascular ischemic diseases.

GIANT PRERETINAL MEMBRANE FORMATION BEHIND A SILICONE OIL BUBBLE IN A HYPOTENSIVE EYE

A patient who suffered a severe contusion injury with scleral rupture and subsequent peracute development of proliferative vitreoretinopathy was referred after wound closure at the local ophthalmology center. Initial treatment of the almost blind eye consisted of vitrectomy, silicone oil tamponade, and intraocular daunorubicin. A vitreous aspirate obtained during surgery was analyzed biochemically by electrophoresis and Western blotting. After a few days, the patient needed further surgical intervention because silicone oil was leaking through the primary sutured scleral wound. A giant fibrinous membrane extending over the posterior pole was removed. Double-label immunofluorescence examination of this specimen showed a positive reaction for transferrin, the cell surface transferrin receptor, fibrinogen, fibronectin, macrophages, and vimentin. No staining was obtained for proliferating cell antigen and T-lymphocyte antigen. The analysis of this case with a very well-defined clinical course offers valuable insight into the early stages of proliferative vitreoretinopathy.