Endothelial Growth Medium Research Articles

Plasma treatment in conjunction with EGM-2 medium increases endothelial and osteogenic marker expressions of bone marrow mesenchymal stem cells

For many tissue engineering applications, an important goal is to create functional tissues in vitro. In order to make such tissues viable, they have to be vascularized. Bone marrow mesenchymal stem cells (BMSCs) are a promising candidate for vascularization. Growth supplements are commonly used to induce BMSCs differentiation, and further improvements in differentiation conditions can be achieved by modifying the cells’ growth environment, such as pretreating the culture dish with gas plasma to modify the surface functional groups that the cells are seeded on. In this work, we compare the effects of different gas plasmas on the growth and differentiation of BMSCs. We treat the growth dish with different plasmas (CO2, N2, and O2) and induce BMSC differentiation on them with endothelial growth medium-2 (EGM-2). We find that EGM-2 by itself does not have strong effect on endothelial differentiation. However, we find that plasma treatment markedly increases the mRNA expression of endothelial cell markers. Interestingly, we also find that the upregulation of osteogenic markers. The data show that plasma treatment in conjunction with EGM-2 can enhance BMSCs differentiation into both endothelial-like cells and osteogenic-like cells. The effects of EGM-2 on BMSCs differentiation are influenced by the plasma-modified surface of the substrate. Our findings provide a method to enhance EGM-2-based cell differentiation, with consequences for tissue engineering and stem cell biology applications.

Neuropilin Controls Endothelial Differentiation by Mesenchymal Stem Cells From the Periodontal Ligament.

Periodontal ligament (PDL) has been reported to be a source of mesenchymal stem cells (MSCs).New vascular networks from undifferentiated cells are essential for repair/regeneration of specialized tissues, including PDL. The current study aims to determine potential of CD105(+)-enriched cell subsets of periodontal ligament cells (PDLSCs) to differentiate into endothelial cell (EC)-like cells and to give insights into the mechanism involved. CD105(+)-enriched PDLSCs were induced to EC differentiation by endothelial growth medium 2 (EGM-2) for 3, 7, 14, and 21 days, with mRNA/protein levels and functional activity assessed by: 1) real-time polymerase chain reaction; 2) Western blotting; 3) fluorescence-activated cell sorting; 4) immunohistochemistry; 5) immunofluorescence; 6) matrigel; and 7) small interfering RNA assays. Data analyses demonstrated that EGM-2 treated PDLSCs presented increased expression of EC markers, including: 1) CD105; 2) kinase domain-containing receptor; and 3) Ulex europaeus agglutinin 1, and were able to form cord/tube-like structures. Gene and protein expression analysis showed that neuropilin 2 (NRP2), a key factor for vascular development, was significantly downregulated during EC differentiation. NRP2 was constitutively expressed in mouse PDL tissues by immunohistochemistry analysis, and NRP2 knockdown in CD105(+)-enriched PDLSCs resulted in increased cord/tube-like structures in a matrigel assay. These findings demonstrated the potential of CD105(+)-enriched PDLSCs to support angiogenesis, and NRP2 as a pivotal factor regulating this process.

An intriguing relation between periodontal and cardiovascular diseases

Periodontitis is a chronic inflammatory condition promoted by bacterial colonization of the gingiva that causes alveolar bone and tooth loss. Under these conditions, bacteria, as well as bacterial products and inflammatory mediators can move from the gingival pocket to the well vascularised periodontal tissues and into the circulation. In fact, recently a possible connection between oral infection and cardiovascular disease was suggested (1). One of the major pathogens involved in the progression of periodontal disease is the Porphyromonas gingivalis (LPS-G). The aim of the study was to induce endothelial differentiation in human periodontal ligaments stem cells (hPDLSCs) (2) on decellularized pig heart valve as scaffold and evaluate the role of LPS-G on cell cultures in terms of reactive oxygen species (ROS) production and NFKB pathway. Many studies have shown that ROS provoked oxidative stress plays a critical role in the development of cardiovascular disease. Excessive generation of ROS can cause cellular dysfunction and injury by directly oxidizing and damaging proteins, DNA and lipids, which ultimately result in cell death. To induce endothelial differentiation, human periodontal ligament stem cells (hPDLSCs) were cultured with endothelial growth medium (EGM-2MV) supplemented with vascular endothelial growth factor (VEGF) and seeded on decellularized pig heart valve. Valve leaflets were incubated for 30 min with 10 μM DCFH-DA at 37°C in humidified incubator. At the end of loading, the valve leaflets were observed using multiphoton microscope. Results of time lapse experiments revealed that after treatment, with 2,5 mg/ml LPS-G, a rapid sustained increase in ROS generation was observed mainly in differentiated hPDLSCs. Comparing the average response of undifferentiated and differentiated hPDLSCs is clearly evident that the latter showed an six fold increase in ROS production after LPS-G exposure, while on undifferentiated appeared to be negligible. Moreover, in endothelial differentiated cells the NFKB nuclear translocation in presence of LPS-G was evident. A reasonable conclusion could be that the treatment of periodontal disease not only improve dental but presumably also cardiovascular health.

Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro

Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

IntroductionDuring endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo.MethodsHuman MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson’s trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo.ResultsPre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups.ConclusionsTaken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

Effect of culture medium on propagation and phenotype of corneal stroma–derived stem cells

Background aimsThe limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. MethodsPrimary human corneal stroma–derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. ResultsAll media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. ConclusionsCulture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.

Fibroblast-Negative CD34-Negative Cells from Human Adipose Tissue Contain Mesodermal Precursors for Endothelial and Mesenchymal Cells.

There is considerable evidence that stem/progenitor cells reside in the vasculature during the prenatal and postnatal stages. The stromal vascular fraction (SVF) of human adipose tissue is markedly rich in blood vessels, and it is a source of mesenchymal/stromal cells (MSCs). Therefore, we hypothesized that, in addition to MSCs, the SVF may contain other mesodermal precursors. However, the SVF has a high content of CD34(+) cells with high proliferative capacity, which can prevent the growth of the most quiescent cells. By using an antifibroblast (FIB) antibody coupled to microbeads, we show that ∼ 90%-95% of the nonhematopoietic CD34(+) cells were retained in the CD45(-)FIB(+) fraction. Reverse transcription-polymerase chain reaction analysis revealed that the CD45(-)FIB(-)CD34(-) cell fraction expressed higher mRNA levels of KDR and GATA2 than its complementary CD45(-)FIB(-)CD34(+) cell fraction, which contained the SVF endothelial cells. Surprisingly, when CD45(-)FIB(-)CD34(-) cells were cultured in endothelial growth medium, they gave rise to endothelial colonies and mesenchymal colonies. Moreover, when CD45(-)FIB(-)CD34(-) cells were cultured in embryonic stem cell expansion medium, they gave rise to cells exhibiting the full range of phenotypes observed in the freshly isolated SVF, including CD34(+) and CD31(+) cells. Together, these results suggest that the CD45(-)FIB(-)CD34(-) cells within the SVF of human adipose tissue function as mesodermal precursors of mesenchymal and endothelial cells.

Transplanted endothelial progenitor cell improves lung structure in neonatal rats exposed to hyperoxia

Objective To study the effect of transplanted endothelial progenitor cell (EPC) on hyperoxia-induced lung injury in neonatal rats. Methods Rat bone marrow mononuclear cells were cultured in endothelial cell growth medium to obtain EPCs, which were identified by morphology, phagocytosis and CD34+ analyses. Sixty neonatal Sprague-Dawley rats were allowed to acclimate in room air for 24 h after birth, and were then divided into four groups (15 per group), including the air group, the hyperoxia group, the EPCs transplantation group and the Nω-nitro-L-arginine methyl ester (L-NAME) intervention group. Neoborn rats in the Air and Hyperoxia groups were fed in the room air or hyperoxia (85% oxygen) for 28 days. For rats in transplantation group were exposed continuously to hyperoxia for 28 days, and got an EPC (1×105 cells) injection on the 21st day. Rats in Intervention group were exposed continuously to hyperoxia for 28 days, got an EPC (1×105 cells) injection on the 21st day, and a daily injection of L-NAME from day 21 to day 28, with a daily dose of 20 mg/kg. Levels of circulating CD34+ cells and serum VEGF expression were detected. Specimens from lung tissues were analyzed by immunohistochemistry or immunofluorescence. The expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and eNOS were detected by real-time polymerase chain reaction and Western-blotting. NO production were detected by nitrate reductase assay. One-way ANOVA and Bonferroni test were used for statistical analysis. Results (1) The cultured cells had a typical cobblestone appearance; double positive cell binding of fluorescein Ulex Europaeus agglutinin-1 and uptake of Dil-labeled acetylated low density lipoprotein accounted for approximately 85% of the total number of cells. CD34+ cells accounted for 68.2%-72.4% of total cultured cells. (2) Circulating CD34+ cells in the air group, hyperoxia group, EPC transplantation group and L-NAME intervention group were (1.91±0.34)%, (1.06±0.10)%, (1.47±0.06)% and (0.77±0.11)% (F=32.710, P=0.000). The number of circulating CD34+ cells in the hyperoxia group was lower than the air group, in the EPC transplantation group the number of these cells was higher than the hyperoxia group, and in the L-NAME intervention group the number of these cells was lower than that in the EPC transplantation group, and the differences between these two groups were statistically significant (P<0.05, respectively). Serum VEGF in the four groups was (7.90±2.72), (6.38±0.72), (14.00±1.66) and (11.70±1.91) pg/ml, respectively. The difference between the four groups was statistically significant (F=22.809, P=0.000), and serum VEGF in the EPC transplantation group was higher than that in the hyperoxia group (P<0.05). (3) Transplanted EPCs could engraft in pulmonary vascular endothelium and alveolar interstitium, and L-NAME intervention significantly reduced the engraftment of EPCs in the lungs (10.7±0.47 / field vs 16.95±0.5 /field, t=17.820, P=0.000). (4) There were significant differences in the radial alveolar count (RAC) and number of microvessels between the four groups (F=859.580 or 211.150, P=0.000, respectively). RAC and the number of microvessels in the hyperoxia group were less than those in the air group (7.98±0.23 vs 13.12±0.20, 3.98±0.42 vs 9.50±0.22, P<0.05, respectively). The number of microvessels in the EPC transplantation group was 5.40±0.41, being higher than that in the hyperoxia group (P<0.05). (5) VEGF mRNA in lungs in the hyperoxia group was lower than that in the air group (0.23±0.16 vs 1.05±0.33, P<0.05); in the EPC transplantation group, VEGF mRNA was higher than that in the hyperoxia group (0.69±0.09 vs 0.23±0.16, P<0.05); and in the L-NAME intervention group, VEGF mRNA was lower than that in the EPC transplantation group (0.31±0.08 vs 0.69±0.09, P<0.05). VEGF protein in the lungs in the hyperoxia group was lower than the air group (0.52±0.01 vs 0.82±0.01, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.58±0.05 vs 0.52±2501, P<0.05). VEGFR2 mRNA in the hyperoxia group was lower than the air group (0.35±0.13 vs 1.07±0.45, P<0.05). eNOS mRNA in the hyperoxia group was lower than the air group (0.46±0.10 vs 1.05±0.36, P<0.05). eNOS protein in the hyperoxia group was lower than the air group (0.32±0.01 vs 0.51±0.03, P<0.05), and was higher in the EPC transplantation group than the hyperoxia group (0.86±0.02 vs 0.32±0.01, P<0.05). Conclusion Transplanted EPC can engraft in the lung tissue, improving alveolar and pulmonary vascular development, which may be associated with upregulation of the expression of eNOS and VEGF in lung. Key words: Endothelial progenitor cells; Stem cell transplantation; Bronchopulmonary dysplasia; Vascular endothelial growth factor A; Nitric Oxide Synthase; Rats

Abstract 614: 1,25-dihydroxyvitamin D Enhances Vegf-stimulated Porcine Adipose-derived Mesenchymal Stem Cells Toward the Endothelial Phenotype Involving Wnt/β-catenin Pathway

Background: Cell-based therapy using adipose-derived mesenchymal stem cells (AMSCs) is an attractive option for re-endothelialization post-angioplasty procedures. Vitamin D levels in circulating blood may regulate AMSC-based re-endothelialization of injured arteries. To our knowledge, the role of vitamin D and vascular endothelial growth factor (VEGF) in the differentiation of adipose-derived AMSCs into endothelial cells (ECs) has not been examined. In this study, we investigated the effect of vitamin D on VEGF-induced differentiation of MSCs into ECs. Methods and Results: MSCs isolated from porcine adipose tissue were CD11b-CD34-CD44+CD73+CD90+ and showed characteristics of MSCs. The MSCs were stimulated and differentiated into ECs with endothelial growth media (EGM+50ng/ml of VEGF) and EGM media containing 10nM of calcitriol (EGM+50ng/ml of VEGF +10nM calcitriol) for 10 days. The EC differentiation was assessed by mRNA expression by qPCR and protein expression by flow cytometry for endothelial cell markers. Calcitriol enhanced EGM+VEGF-induced differentiation of MSCs into ECs, as revealed by 2-fold increase in mRNA and 4-fold increase in protein expression of EC markers. Angiogenesis assay and acetylated low density lipoprotein (LDL) uptake assay were used to assess the endothelial functionality that showed significant increase in capillary tube sprouting, and an increase in LDL uptake by the differentiated cells in response to EGM +VEGF+ calcitriol. Examination of the findings from Wnt Pathway array revealed a 3-fold decrease in β-catenin and 3-fold increase in KREMEN1 protein in the cells treated with EGM + VEGF+calcitriol. Conclusion: Vitamin D significantly enhanced VEGF-induced differentiation of MSCs into endothelial cells. Thus, vitamin D status of the patients undergoing coronary intervention might regulate the development of thrombosis and intimal hyperplasia.

Enhancement of endothelial differentiation of adipose derived mesenchymal stem cells by a three-dimensional culture system of microwell

Adipose derived mesenchymal stem cells (AdMSCs) have been demonstrated to have ability to differentiate into several cell lineages, including endothelial cells. The low endothelial differentiation efficiency, however, limits further clinical application of AdMSCs for therapeutic angiogenesis. This study was designed to investigate the feasibility to promote endothelial differentiation efficacy of AdMSCs using microwell array as a 3-D culture system. AdMSCs aggregates were prepared using photocrosslinkable polyethylene glycol dimethacrylate (PEGDM) derived microwell. AdMSCs aggregated and formed well defined 3-D aggregates following seeding. The microwell was effective in regulating the size of AdMSCs aggregates with low variation. AdMSCs within the 3-D aggregates maintained the cell surface epitopes of AdMSCs with high viability. Endothelial growth medium was used to induce the in vitro endothelial differentiation of AdMSCs. Both gene expression results from real time PCR and protein expression data from immunofluorescent staining revealed that 3-D cultured aggregates significantly promote the endothelial differentiation efficacy of AdMSCs. AdMSCs or AdMSCs aggregates were injected into the subcutaneous space of nu/nu mice to investigate the endothelial differentiation in vivo. The immunofluorescent staining data indicated promoted endothelial differentiation of 3-D aggregates compared with 2-D AdMSCs. Aggregates dissociated cells were obtained by transferring 3-D aggregates onto the adherent surfaces. Cells dissociated from induced aggregates were still positive for endothelial specific markers and were able to form endothelial-like tube structures on matrigel, indicating the endothelial properties. We conclude that microwell is an ideal 3-D culture system for promoting endothelial differentiation efficacy of AdMSCs.

Characterization of endothelial colony-forming cells from peripheral blood samples of adult horses

To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. Jugular venous blood samples from 24 adult horses. Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

Optimization of in vitro cultivation strategies for human adipocyte derived stem cells

With adipose-derived stem cells being in the focus of research in regenerative medicine, the need arises for fast reliable cultivation protocols. We have tested the cultivation of human adipose-derived stem cells in endothelial cell growth medium prior to induction and differentiation, against the long-established use of DMEM/F12 medium-based cultivation protocols. We found that cultivation in endothelial cell growth medium not only accelerates growth before induction and differentiation, but also allows shorter induction and differentiation times than those following precultivation with DMEM/F12 medium with regard to the formation of mature adipocytes and to the viability undifferentiated cells. These results were first observed morphologically but could be confirmed by performing adiponectin ELISA and cell proliferation assays.

Expression and Characterization of Antimicrobial Peptide LL-37 in Dog Peripheral Blood Endothelial Progenitor Cells In Vitro

This study aimed to construct antimicrobial peptide LL-37 expressed lentiviral vector and explore the expression of LL-37 in endothelial progenitor cells (EPCs) from dog periphery mononuclear cells (PBMCs). The methodology on obtaining EPCs from dog PBMCs was explored and infection approach about the expression of LL-37 in EPCs was optimized. LL-37 was amplified, digested by Age I and constructed into pGC-FU vector. Meanwhile, EPCs were generated from PBMCs using endothelial growth medium. Markers including acetylated low density lipoproteins (Dilac-LDL), Ulex lectin (UEA-1), von Willebrand factor (vWF), VE-cadherin, CD34 and CD133 were used to identify EPCs. Then, LL-37 was introduced into EPCs using lentivirus infection with green fluorescent protein (GFP) as the biomarker. Finally, we used western-blot to examine the expression of LL-37 in EPCs. The pGC-FU-LL-37-GFP was successfully constructed. Viral particles existed in 10−4 μL group with titer of 2 × 108 TU/mL. Both early (round-shaped morphology) and late (pebble-shaped appearance) EPCs were observed. And these EPCs were confirmed by Dil-acLDL uptake and FITC-UEA-1 binding. An approximate 50 % of infection efficacy was confirmed by immunofluorescence. Furthermore, we successfully observed LL-37 expression in EPCs via western blot analysis. In conclusions, LL-37 expressed in the EPCs was successfully achieved. However, the anti-infection and angiogenesis of LL-37 expressed EPCs need further research in future.

Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

Systemic arterial hypertension (SAH), a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs), which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2) increases their therapeutic effects in spontaneously hypertensive rats (SHRs). Adult female SHRs were administered an intraperitoneal injection of vehicle solution (n = 10), MSCs cultured in conventional medium (DMEM plus 10% FBS, n = 11), or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n = 10). Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days) in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH.

Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling.

Neonatal hypoxic-ischemic (HI) brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs) from adipose-derived stem cells (ASCs) and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1) and vascular endothelial growth factor receptor 2 (VEGFR2) was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

Ox-LDL induces dysfunction of endothelial progenitor cells via activation of NF-κB.

Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity. Endothelial progenitor cells (EPCs) are thought to contribute to endothelial recovery after arterial injury. Oxidized low-density lipoprotein (ox-LDL) can induce EPC dysfunction, but the underlying mechanism is not well understood. Human EPCs were cultured in endothelial growth medium supplemented with VEGF (10 ng/mL) and bFGF (10 ng/mL). The cells were treated with ox-LDL (50 µg/mL). EPC proliferation was assayed by using CCK8 kits. Expression and translocation of nuclear factor-kabba B (NF-κB) were evaluated. The level of reactive oxygen species (ROS) in cells was measured using H2DCF-DA as a fluorescence probe. The activity of NADPH oxidase activity was determined by colorimetric assay. Ox-LDL significantly decreased the proliferation, migration, and adhesion capacity of EPCs, while significantly increased ROS production and NADPH oxidase expression. Ox-LDL induced NF-κB P65 mRNA expression and translocation in EPCs. Thus ox-LDL can induce EPC dysfunction at least by increasing expression and translocation of NF-κB P65 and NADPH oxidase activity, which represents a new mechanism of lipidemia-induced vascular injury.

Human Embryonic Stem Cell Lines BJNhem 19 and 20 Fail to Differentiate Into Lung Lineage Specific Cells despite Induction through Guided Endodermal Differentiation

Regenerative Therapy’s first and most critical requisite is the ex vivo synthesis of fully functional desired cells to replenish those lost by the body in a degenerative disease. The etiology of degeneration differs from disease to disease, likewise, strategies to regenerate lost tissue in its heterogeneousness, should also cater to the regeneration process and to customize the same, the researcher ought to devise strategies to repair or replace or regenerate in the appropriate spatio-temporal format. For example, in the inflammation-degeneration-induced pathophysiological situations of the respiratory epithelium, human embryonic stem cells have been used in a tissue engineering format to induce differentiation and amplification into the desired type of cell, in this case, the nonciliated squamous epithelial cells. To this end, we were given two human embryonic stem cell (hESC) lines and the main objective of our research work was to get BJNhem19 and BJNhem20 human embryonic stem cell (hESC) lines to differentiate into lung epithelial lineage–specific cells (i.e. alveolar epithelial type I and type II cells and clara cells) which are the key cells to degenerate in most degenerative lung ailments. This in order to generate a potentially unlimited supply of cells of the desired phenotype for use in a novel cell based therapy to repair lung injury. The strategy was to use guided endodermal differentiation by direct administration of one or more growth factors known to be involved in lung development in 2D cell cultures and characterize the cells for the desired markers. According to a tried strategy, the undifferentiated hESC were taken through embryoid body formation and then subjected to induction by defined growth factors in small airways growth medium and bronchiolar endothelial growth medium. Cells could not be grown feeder-free. Attempts to aid growth with combinations of extra cellular matrix plus defined medium (GeltrexR) as well as enriched media such as Matrigel and mTeSR1 did not yield satisfactory result. When grown on feeders also cell growth was again not optimum and practically none differentiated appreciably into the desired phenotype and maintained their pluripotent characters. After 5 days in induction media, they displayed fibroblast like features characterized by FACS. If cell lines are thus non-response to specific and guided endodermal induced differentiation, the obtained data is animportant information for cell repositories as numerous research labs screen various cell lines of embryonic origin with the specific aim to induce differentiation into a desired phenotype for functional translation into regenerative therapy. This study therefore fills an existing lacuna in available information regarding behavior of these two hESC available for work to the scientific world in several stem cell banks and shall prevent unnecessary and redundant further screening and save valuable resources.

Application of Adult Stem Cells in Medicine.

Application of Adult Stem Cells in Medicine.

Heterogeneity in Human Embryonic Stem Cells May Prevent Endodermal Guided Differentiation

This study aims at exploring the use and peculiarities of stem cell therapy for pulmonary injury and degeneration. The success of stem cell therapy for clinical use needs the validation of several issues. Regenerative Therapy’s first and most critical requisite is the ex vivo synthesis of desirable cells to replenish those lost by the body in a degenerative disease. The etiology of degeneration differs from disease to disease, likewise, strategies to regenerate lost tissue in its heterogeneousness, should also cater to the regeneration process and to customize the same, the researcher ought to devise strategies to repair or replace or regenerate in the appropriate spatio-temporal format. For example, in the inflammation-degeneration-induced pathophysiological situations of the respiratory epithelium, human embryonic stem cells have been used in a tissue engineering format to induce differentiation and amplification into the desired type of cell, in this case, the non-ciliated squamous epithelial cells. To this end, we were given two human embryonic stem cell (hESC) lines and the main objective of our research work was to get BJNhem19 and BJNhem20 human embryonic stem cell (hESC) lines to differentiate into lung epithelial lineage–specific cells (i.e. alveolar epithelial type I and type II cells and clara cells) which are the key cells to degenerate in most degenerative lung ailments. This in order to generate a potentially unlimited supply of cells of the desired phenotype for use in a novel cell based therapy to repair lung injury. The strategy was to use guided endodermal differentiation by direct administration of one or more growth factors known to be involved in lung development in 2D cell cultures and characterize the cells for the desired markers. According to a tried strategy, the undifferentiated hESC were taken through embryoid body formation and then subjected to induction by defined growth factors in small airways growth medium and bronchiolar endothelial growth medium. Results were not satisfactory and the cells showed poor growth and practically none differentiated appreciably let alone into the desired phenotype. This non-response to induced differentiation is important information as numerous research labs screen various cell lines of embryonic origin with the specific aim to induce differentiation into one or more desired phenotype for functional translation into regenerative therapy. This study therefore fills an existing lacuna in available information regarding behavior of these two hESC available for work to the scientific world in several stem cell banks and shall prevent unnecessary and redundant further screening and save valuable resources.