EGM-2 Medium Research Articles

Abstract 159: Vascularized 3D cancer organoid research platform using surface acoustic waves

Abstract Recently, organoids have been widely used in cancer research. However, cancer organoids have limitation in reflecting physicochemical microenvironments of tumor. In particular, vascular networks not only provide oxygen and nutrients to the tumor, but also play an important role as a channel through which drugs move in cancer. Vascularization occurs in an uncontrollable form and speed in conventional methods. Here, we regulate the directionality of the vascular bed using the surface acoustic waves(SAW) technology by which, vascular cells are locally aligned. Aligned cells exhibited an improved, cell-cell contact enhancing the vascular system maturation in a couple days. Organoids are constructed from gastric cancer patients with four molecular subtypes according to TCGA. Organoids were dissociated into a single cell to make a co-culture spheroid with HUVEC cells. Co-cultured spheroids were formed by adding fibronectin according to the characteristics of the patient sample. The SAW device is built with interdigitated transducer, NiNbO3 substrate, polydimethylsiloxane(PDMS) and coverglass. After putting co-culture spheroids and HUVEC cells mixed with fibrin gel into the device, patterning is performed using surface acoustic waves with shaking in EGM-2 media. After fixing the models, CD31 and F-actin are stained and imaged through immunofluorescent. Immunohistochemistry was performed to confirm the blood vessel cross section. The blood vessels patterned with SAW were well aligned in a straight line, and a lumen structure was formed in a couple days. When co-cultured spheroids were used compared to mono-cultured spheroids, the interaction with external HUVECs was improved, allowing for efficient vascular organism models. As evidence for this, it was confirmed that HUVEC inside the co-cultured spheroid and HUVEC outside were connected through interaction when PKH dyeing was done in a different color. We showed that blood vessels are formed faster and thicker in patterning models than in random models, and interactions with co-cultured spheroids are also high. Through immunofluorescent staining, we found that the number of vascular branches linked to spheroids was higher in the SAW model. In addition, in the H&E results, it was confirmed that the blood vessels were arranged in a line, and that differentiation occurred appropriately to form a tubular structure. In addition, the number of blood formed inside the spheroid was higher. Currently, this model is used to test anti-cancer efficacy using FGFR inhibitors and ramucirumab in FGFR2-amplified patient-derived organoids. An efficient vascularized 3D cancer organoid model was constructed using acoustic waves. Our vascularized 3D cancer organoid research platform will be applied for specific drug screening platform that directly aid the treatment of the specific patients with various drugs including chemoagents, targeted drugs, and immune checkpoints inhibitors. Citation Format: Hyoyoung Kim, Yunam Lee, Byungjun Kang, Un-Jung Yun, Yunjung Choi, So Hui Kim, Hyunki Kim, Hyungsuk Lee, Minkyu Jung. Vascularized 3D cancer organoid research platform using surface acoustic waves [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 159.

Infusion of Cultured Endothelial Progenitor Cells Boosts Hematopoietic Recovery and Increases the Expression of LepR+ Cells after Irradiation

The hemopoietic system's function of producing blood cells and immune cells during thorough life relies on the Maintainance and differentiation of hematopoietic stem cells (HSCs). HSCs activities were tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow. However, during the routine treatment of hematopoietic diseases, such as chemotherapy and radiotherapy, the niche was inevitably impaired which is one of the essential causes of hematopoietic suppression. Our previous research results showed that infusion of cultured endothelial progenitor cells (EPCs) could boost hematopoietic recovery after HSCT. And the latent mechanisms could be that EPCs can differentiate into endothelial cells (ECs) in vivo. However, the roles of EPCs play in the bone marrow niche and the specific mechanism remain unknown. Here we designed further to investigate the effect and mechanism of EPCs in boosting bone marrow microenvironment recovery after total body irradiation (TBI). This may offer a new cue for therapy of hematopoietic suppression after the treatment of malignant hematological diseases. Bone marrow-derived Primary EPCs isolation and culture were described as before. Briefly, Bone marrow cells from Balb/c or C57BL/6J mice aged 6-8 weeks were cultured with EGM-2 medium which was changed in 36 h, the adherent cells formed colonies identified as elongated sprouting cells radiating from a central core of round cells and identified as VEGFR2+; CD133+; CD144+; SCA1+ by FACS on days 4-6. To assess the effects of the EPCs in boosting hematopoiesis, Balb/c mice underwent TBI with a lethal dose and were injected withEPCs or the same volume of PBS via tail vein in 4-6 hours. The survival of the EPC group was elevated ( survival: 8/9 vs 0/13; p＜0.001). C57BL/6J mice underwent TBI with a sublethal dose and were injected with the cultured EPCs isolated from C57BL/6-Tg(CAG-EGFP) mice or the same volume of PBS via tail vein in 4-6 hours. Results showed that EPCs boost the recovery of hematopoietic cells in the bone marrow and peripheral blood cells; Immunofluence (IF) staining showed that Infused EPCs with GFP fluorescence were home to bone marrow and differentiated into ECs which were marked with endomucin (EMCN); Moreover, assessments of the alteration of bone marrow niche using H&E staining revealed that infusion of EPCs reduced the accumulation of fat and using multi-analyte flow assay showed that mice infused with EPCs express a higher level of M-CSF and TGF-β in plasma while the level of SCF and IL-6 both in plasma and bone marrow supernatant were lower compared to TBI. Furthermore, to assess the change of sinusoidal niche, we used IF staining and found that the sinusoids were dilated and perisinusoidal stromal cells identified as LepR+ were massively lost on the 7th day after TBI while the dilated sinusoidal lumens were narrowed (11.05±1.789 vs 20.86±4.573，p=0.0258) and LepR+ cells were upregulated on the 21st day after the infusion of EPCs which indicated that the sinusoidal niche has a better recovery compared to TBI. In Conclusion, EPCs could differentiate into endothelial cells in vivo boosting the recovery of the hematopoietic system. And the potential mechanism is that EPCs repair the bone marrow sinusoidal niche damaged by irradiation.

Abstract P3043: Functional Role Of Endothelial Mitochondrial Oxidative Stress In Cognitive Impairment: Effects Of L-arginine In Hypertensive Frail Older Adults

Background: Hypertension is common in older adults, particularly in frailty. The association of hypertension and frailty leads to a high-risk of cognitive impairment in older adults. Endothelial dysfunction has been shown to underlie both hypertension and cognitive impairment. L-Arginine is an amino acid involved in many biological processes and is a substrate of two enzymes: nitric oxide (NO) synthase (NOS) and arginase (NOA). L-Arginine is fundamental for NO production by endothelial cells, regulating vascular tone and cardiovascular homeostasis, improving inflammation and oxidative stress. On these grounds, we hypothesized that L-Arginine could improve cognitive impairment via its effect on endothelial mitochondria. Methods: To assess the effects of L-Arginine on endothelial oxidative stress and cognitive function, we used an in vitro and a clinical approach. First, we treated with L-Arginine human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in EGM-2 medium and incubated at 37°C and 5% CO 2 . HUVECs were plated on glass bottom culture dishes and treated with Angiotensin II (1 μM) and Angiotensin II with L-Arginine (500 μM) in EGM-2 medium for 24h. The generation of reactive oxygen species (ROS) was quantified using MitoSOX™ Red (an established ROS probe that accumulates in mitochondria), incubating cells for 10 min at 37°C and 5% CO 2 , as we previously described. We then designed a single-blind, placebo-controlled, parallel-group, randomized trial to observe the effects of 4-weeks oral supplementation of L-Arginine on global cognitive function of hypertensive frail older patients. Results: In vitro , we demonstrated that L-Arginine significantly attenuated Angiotensin II-induced oxidative stress in endothelial cells. Co-treatment of HUVECs with Ang II and L-Arginine significantly attenuated mitochondrial ROS production (p<0.001). In the clinical study, we evaluated 35 frail hypertensive elderly patients assigned to L-Arginine and 37 assigned to placebo: at follow-up, we found a significantly improved Montreal Cognitive Assessment (MoCA) test score in the L-Arginine treated arm compared to placebo (p<0.05). Conclusions: Taken together, our findings indicate for the first time that oral L-Arginine supplementation improves cognitive impairment in frail hypertensive older adults through a mechanism that involves endothelial mitochondrial ROS.

Up-regulation of GLP-1R improved the dysfunction of late EPCs under hyperglycemia by regulating SIRT1 expression

The dysfunction of endothelial progenitor cells (EPCs) is closely associated with diabetic vascular complications. Both glucagonlike peptide-1 receptor (GLP-1R) and silent information regulator 1 (SIRT1) can control systemic glucose homeostasis and protect endothelial cells against hyperglycemia-induced oxidative stress. In this study, we mainly assessed the role played by SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation. Human peripheral blood mononuclear cells (PBMCs) were cultured in EGM-2 medium and induced to differentiate into EPCs and 25 mM glucose was used to stimulate EPCs to obtain a hyperglycemia condition. Subsequently, the expression and location of GLP-1R and SIRT1 in EPCs were detected. After GLP-1R or SIRT1 knockdown, or the treatment by GLP-1R agonist and/or SIRT1 agonist/inhibitor, the effects of SIRT1 and GLP-1R and their relationship in regulating the function of late EPCs under hyperglycemia stimulation was studied by detecting the apoptosis, migration, adhesion and angiogenicity abilities of EPCs. Results demonstrated that, in high-glucose stimulated EPCs, the expression of GLP-1R and SIRT1 was down-regulated. The knockdown of either GLP-1R or SIRT1 could increase EPCs apoptosis and weaken the migration, adhesion and angiogenicity abilities of EPCs. In addition, the improvement effects of Exendin-4 or GLP-1R over-expression on EPCs dysfunction could be weakened to some degree under SIRT1 knockdown. In conclusion, both GLP-1R and SIRT1 expression played important roles in regulating EPCs dysfunction under hyperglycemia and the up-regulation of GLP-1R improved the dysfunction of late EPCs by regulating SIRT1 expression.

An optimal non-viral gene transfer method for genetically modifying porcine bone marrow-derived endothelial progenitor cells for experimental therapeutics.

No currently available treatment is able to generate new contractile tissue or significantly improve cardiac function after myocardial infarction (MI), a leading cause of morbidity and mortality worldwide. Although gene transfer-enhanced endothelial progenitor cells (GTE-EPCs) show effectiveness in MI treatment in small animal models, no clinical trials using GTE-EPCs have been documented. Before the introduction of GTE-EPCs into human trials, gene-transfer-mediated augmentation of EPC function in animal models that reflect the human MI scenario should be tested. In this regard, a porcine model is the best choice since pigs have cardiac size, hemodynamics and coronary anatomy similar to that of humans. To examine GTE-EPC therapeutic efficacy in pig MI models, an efficient method for gene transfer into pig EPCs is required, which however, has been poorly documented. Pig bone marrow mononuclear cells were isolated and cultured in EGM-2 medium to obtain bone marrow-derived EPCs (BM-EPCs) that were characterized by immunostaining and the tube formation assay. Gene transfer was optimized in 6-well plates using a GFP and a VEGF plasmid, and scaled up in T75 flasks. Gene transfer efficiency was determined by fluorescence microscopy and flow cytometry. VEGF levels were measured by ELISA. Cell proliferation was assayed by the CCK-8 kit. (1) BM-EPCs expressed VEGFR2 and eNOS but not CD45 protein, and formed tube structures on Matrigel; (2) several chemical compounds were explored with the highest transfection efficiency of 41.4% ± 5.8% achieved using Lipofectamine 3000; (3) the VEGF level in culture medium after VEGF transfection was 378 ± 48 ng/106 cells; and (4) BM-EPCs overexpressing VEGF had significantly enhanced proliferation than GFP-transfected EPCs. A simple, easy and cheap method that can be applied to produce a large number of genetically-modified BM-EPCs was established, which will facilitate the study of GTE-EPC therapeutic efficacy in pig MI model.

Influence of culture media on the derivation and phenotype of fetal-derived placental mesenchymal stem/stromal cells across gestation

IntroductionDerivation of pure fetal placental mesenchymal stem/stromal cells (pMSCs) is key to understand their role in placental development. However, isolated pMSCs are often contaminated by maternal-derived decidual MSCs (dMSCs). EGM-2 medium promotes the derivation of term fetal pMSCs, but the extent of first-trimester maternal pMSC contamination remains unclear. Culture media can also affect MSC phenotype. Here, we examined the effects of culture media on maternal pMSC contamination and fetal pMSC phenotype across gestation. MethodspMSCs were derived from first-trimester or term placentae in advanced-DMEM/F12 medium or EGM-2 medium. Proportions of maternal (XX) and fetal (XY) cells in male pMSC cultures were determined by fluorescence in-situ hybridization. pMSC phenotype was analysed by flow cytometry, immunohistochemistry and Alamar blue proliferation assays. ResultsWhen derived in advanced-DMEM/F12, all first trimester pMSC isolates exhibited maternal contamination (>72% XX cells, n = 5), whilst 7/9 term pMSC isolates were >98% fetal. When derived in EGM-2, all first trimester (n = 4) and term (n = 9) pMSC isolates contained 95–100% fetal cells. Fetal pMSCs in EGM-2 proliferated 2-fold (first-trimester) or 4-fold (term) faster than those in advanced-DMEM/F12 (p < 0.05, n = 3). Fetal pMSCs in both media expressed the generic MSC marker profile (CD90+, CD105+, CD73+, CD31-, CD34-, CD144-). However, pMSCs transferred from EGM-2 to advanced-DMEM/F12 increased expression of smooth muscle cell markers calponin and α-smooth muscle actin, and decreased expression of the vascular cell marker VEGFR2 (n = 3). ConclusionsDeriving first-trimester pMSC in EGM-2 dramatically reduces maternal dMSC contamination. Media affects fetal pMSC phenotype, and careful consideration should be given to application specific culture conditions.

Cardiovascular Effects of Polychlorinated Biphenyls and Their Major Metabolites.

Background:Xenobiotic metabolism is complex, and accounting for bioactivation and detoxification processes of chemicals remains among the most challenging aspects for decision making with in vitro new approach methods data.Objectives:Considering the physiological relevance of human organotypic culture models and their utility for high-throughput screening, we hypothesized that multidimensional chemical-biological profiling of chemicals and their major metabolites is a sensible alternative for the toxicological characterization of parent molecules vs. metabolites in vitro.Methods:In this study, we tested 25 polychlorinated biphenyls (PCBs) [PCB 3, 11, 52, 126, 136, and 153 and their relevant metabolites (hydroxylated, methoxylated, sulfated, and quinone)] in concentration–response () for effects in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs) (iPSC-derived and HUVECs). Functional phenotypic end points included effects on beating parameters and intracellular flux in CMs and inhibition of tubulogenesis in ECs. High-content imaging was used to evaluate cytotoxicity, mitochondrial integrity, and oxidative stress.Results:Data integration of a total of 19 physicochemical descriptors and 36 in vitro phenotypes revealed that chlorination status and metabolite class are strong predictors of the in vitro cardiovascular effects of PCBs. Oxidation of PCBs, especially to di-hydroxylated and quinone metabolites, was associated with the most pronounced effects, whereas sulfation and methoxylation of PCBs resulted in diminished bioactivity.Discussion:Risk characterization analysis showed that although in vitro derived effective concentrations exceeded the levels measured in the general population, risks cannot be ruled out due to the potential for population variability in susceptibility and the need to fill data gaps using read-across approaches. This study demonstrated a strategy for how in vitro data can be used to characterize human health risks from PCBs and their metabolites. https://doi.org/10.1289/EHP7030

Comparison of the effectiveness of available sources of autologous colony-forming endothelial cells

Endothelial Colony-forming cells (ECFCs) are valuable material for tissue vascular engineering and cell therapy of ischemic tissues. Difficult isolation is the main problem for using of ECFCs. ECFCs isolation from peripheral blood and adipose tissue has been previously described. In the presented research we compared effectiveness of peripheral blood, subcutaneous and epicardial adipose tissue for the ECFCs isolation without cell sorting. ECFCs was isolated from peripheral blood, subcutaneous and epicardial adipose tissue obtained from coronary heart disease patients (males, n=8) undergoing elective coronary artery bypass surgery. The stromal-vascular fraction of subcutaneous (SVF-ST) and epicardial (SVF-ET) adipose tissue as well as the mononuclear blood fraction (MNF) were cultivated in the complied EGM-2 medium. Cell cultures phenotyping was performed by flow cytometry and confocal microscopy. Their angiogenic (Matrigel) and proliferative activity (xCELLigence analyzer) in vitro were studied. ECFCs were isolated from MNF in 50% of cases, from SVF-ST in 12.5% and SVF-ET in 37.5%. The proliferative activity of ECFCs isolated from adipose tissue was low while cultures from MNF showed high and medium activity in 75% of cases. Pure ECFCs (more 99%) were obtained from MNF to third passage without cell sorting. Cultures from adipose tissue were contaminated by mesenchymal-stromal cells (MSCs) and contained ECFCs and MSCs. Thus, peripheral blood is the most effective source of autologous ECFCs compared with adipose tissue for this isolation method. However, adipose tissue is a suitable source of MSC and mixed cultures of MSC and endothelial cells.

648 Adipose-derived stem cells in wound healing: comparison with fibroblasts and keratinocytes interaction

Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue. These cells are considered very attractive for their relative abundance and accessibility in the human body. Keratinocytes and fibroblasts cells play important roles in the skin-wound healing process and are the cell types activated by trauma. The aim of our study was focused to evaluate ADSCs ability to build a dermal matrix to be potentially used as a dermal substitute in the field of wound healing. ADSCs were defined as Cluster Differentiation (CD) markers CD90, CD73 and CD105 positive cells and were capable of chondrogenic, osteoblastic and adipogenic differentiation using established protocols. We evaluated several ADSCs abilities in comparison with fibroblasts in order to secrete crucial components of the extracellular matrix such as collagen I and fibronectin. Moreover the culture medium conditioned by ADSCs enhanced the rate of wound closure of fibroblasts and keratinocytes on an in vitro skin wound healing model. Ki67 immunostaining evaluated keratinocyte migrating capacity and colonization on this ADSC-induced matrix demonstrating its ability to promote re-epithelization. Furthermore in the angiogenic 3D assay on Matrigel, ADSCs grown in EGM-2 medium started to form microvessels within 24 hours. This study suggests that ADSCs support vascular network formation in vitro and demonstrates that the ADSCs trophic activity may play an important role in skin wound healing by affecting keratinocytes migration and dermal fibroblasts contribution to generate extracellular matrix.

Effect and Mechanism of Kir2.1 Channel Overexpression on Transdifferentiation of Endothelial Progenitor Cells

Objective The propose of the study is to investigate the specific effects of the mechanically sensitive channel Kir2.1 on the transdifferentiation of EPCs so as to understand the molecular mechanism of pathological vascular remodeling. Methods Endothelial progenitor cells (EPCs) were isolated from rat bone marrow and cultured in EGM2 medium in vitro. The recombinant lentiviral vectors carrying Kir2.1 (NM_017296.1) gene was designed and constructed in order to overexpress the gene. The smooth muscle cells (SMCs) molecules marker on EPCs, such as α-SMA, FSP1 and α-SM22, were detected by RT-PCR and cellular immunofluorescence. In addition, cell angiogenic capacity and migration in vitro were assessed by Matrigel and Transwell methods respectively. Moreover, neointimal thickening was evaluated in the surgery model of balloon injury of rat carotid artery in vivo. Result The results showed that the expression levels of α-SM22, FSP1 and α-SMA were up-regulated in the Kir2.1 overexpression group compared with the control. The number of migrating cells in the Kir2.1 overexpression group was significantly higher than that in the scramble group, while quantitative assessment further confirmed that the Kir2.1 overexpression strongly attenuated the ability of bone marrow-derived EPC to form tube-like structures in Matrigel assay. Compared with the control group, morphometric analysis showed ratio of intimal area/medial area (I/M) in rats was increased in rats transplanted with Lenti-Kir2.1 overexpression. Conclusion It is indicated that the overexpression of channel Kir2.1 induces EPCs transdifferentiated into mesenchymal transition SMCs (EndoMT). It may provide a potential target for the treatment or prevention of pathological vascular remodeling disease.

Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells.

For stem cell differentiation, the microenvironment can play an important role, and hydrogels can provide a three-dimensional microenvironment to allow native cell growth in vitro. A challenge is that the stem cell's differentiation can be influenced by the matrix stiffness. We demonstrate a low-toxicity method to create different stiffness matrices, by using a photopolymerizable gelatin methacrylate (GelMA) hydrogel cross-linked by blue light (440nm). The stiffness and porosity of GelMA hydrogel is easily modified by altering its concentration. We used human bone marrow mesenchymal stem cells (MSCs) as a cell source and cultured the GelMA-encapsulated cells with EGM-2 medium to induce endothelial differentiation. In our GelMA blue light hydrogel system, we found that MSCs can be differentiated into both endothelial-like and osteogenic-like cells. The mRNA expressions of endothelial cell markers CD31, von Willebrand factor, vascular endothelial growth factor receptor-2, and CD34 were significantly increased in softer GelMA hydrogels (7.5% and 10%) compared with stiffer matrices (15% GelMA). On the other hand, the enhancements of osteogenic markers mRNA expressions (Alkaline phosphatase (ALP), Runx2, osteocalcin, and osteopontin) were highest in 10% GelMA. We also found that 10% GelMA hydrogel offered optimal conditions for MSCs to form capillary-like structures. These results suggest that the mechanical properties of the GelMA hydrogel can influence both endothelial and osteogenic differentiation of MSCs and sequent capillary-like formation.

Adipose-Derived Stem Cells Can Contribute to Vascular Network Formation in Poly(ethylene Glycol) Hydrogel Scaffolds

Although adipose-derived stem cells (ADSCs) can influence wound healing, their role in neovascularization is unclear. Utilizing three-dimensional in vitro, we sought to determine whether ADSCs cultured under varying conditions could contribute to vascular network formation, functioning as either endothelial cells (ECs) or supporting pericytes.. To study this, ADSCs were encapsulated in 3D hydrogels either with human brain vascular pericytes (HBVP, to show ADSC functions as ECs) or with human umbilical vein endothelial cells (HUVEC, to show ADSC functions as pericytes). The hydrogel used provides a 3D cell-adhesive, proteolytically degradable cell culture matrix that supports formation of vascular networks by encapsulated endothelial cells and pericytes. For this study, ADSCs were cultured in either basal media (basal) or EGM-2 media with 20 ng/mL of VEGF (stimulated) for 7 days and then encapsulated with HUVECs or HBVPs. When encapsulated with HBVPs, both basal and stimulated ADSCs were capable of forming CD31+ tubule-like networks, indicating endothelial function. In co-culture with HUVECs, basal and stimulated ADSCs were capable of enhancing and stabilizing formation of CD31+ tubule-like networks by the HUVECs and localized along the outer surfaces of the endothelial tubules, indicating pericyte-like function. These studies were repeated with diabetic ADSCs to examine the influence of this phenotype on the cells’ ability to influence neovascularization. Both basal and stimulated diabetic ADSCs were capable of supporting tubule formation by HUVECs, though to a lesser degree than non-diabetic ADSCs. Notably, diabetic ADSCs were not capable of forming CD31+ tubules when co-cultured with HBVPs, indicating loss of endothelial function. Adipose-derived stem cells (ADSCs) are known to play a role in wound healing; however, the exact role of these cells in neovascularization is unclear. Our work focuses on understanding specific cell–cell interactions between ADSCs, endothelial cells, and mesenchymal support cells within a 3D poly(ethylene glycol) hydrogel scaffold. Furthermore, we sought to understand the effect of the diabetic phenotype on this phenomenon as well.

Porcine Neonatal Islet-like Cell Clusters Cultured with EGM2 Medium Enhance their Resistance to Apoptosis in Islet Xenotransplantation

Introduction Pig neonatal islet-like cell clusters (NICC) are an attractive source of insulin-producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood mediated inflammatory reaction remains to be overcome. Materials and Methods EGM2 medium was depleted with hydrocortisone and supplemented with 50 mM isobutylmethlxanthine, 10 mM nicotinamide,10 mM glucose. Modified EGM2 medium was used to culture NICC at day 1, the day after isolation and changed every other day. When NICC cultured with modified EGM2 or control medium Ham’s F-10 at day 7, they were collected to do the following assays. AO/EB staining were used to evaluate the viability of NICC. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon expression was measured by real-time PCR. Serum starvation and Cocl2 treatment was used to simulate the microenvironment post islet transplantation. The viability of NICC was evaluated by FACS and the proposed mechanism was detected by western blotting. Results Compared with Ham’s F-10 medium, culturing NICC with modified EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon and also higher mitochondria function. Moreover, modified EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or CoCl2-induced hypoxia condition than their Ham’s F-10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti-apoptotic Bcl-2 family member Mcl-1. Discussion NICC cultured with EGM2 medium not only improved their viability and mitochondria function but also enhanced their resistance to apoptosis under the hypoxia environment. Conclusion Culturing NICC with modified EGM2 provides a simple and effective approach to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation. This project was supported by NSFC 81201171 and Hunan Provincial Natural Science Foundation of China 2017JJ3423.

Acceleration of diabetic wound healing with adipose-derived stem cells, endothelial-differentiated stem cells, and topical conditioned medium therapy in a swine model

ObjectiveThe purpose of our study was to investigate the effect of adipose-derived stem cells (ASCs), endothelial-differentiated ASCs (EC/ASCs), and various conditioned media (CM) on wound healing in a diabetic swine model. We hypothesized that ASC-based therapies would accelerate wound healing. MethodsDiabetes was induced in four Yorkshire swine through intravenous injection of streptozotocin. ASCs were harvested from flank fat and cultured in either M199 or EGM-2 medium. A duplicate series of seven full-thickness dorsal wounds were surgically created on each swine. The wounds in the cellular treatment group underwent injection of low-dose or high-dose ASCs or EC/ASCs on day 0, with a repeat injection of one half of the initial dose on day 15. Wounds assigned to the topical CM therapy were covered with 2 mL of either serum-free M199 primed by ASCs or human umbilical vein endothelial cells every 3 days. Wounds were assessed at day 0, 10, 15, 20, and 28. The swine were sacrificed on day 28. ImageJ software was used to evaluate the percentage of wound healing. The wounded skin underwent histologic, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay examinations to evaluate markers of angiogenesis and inflammation. ResultsWe found an increase in the percentage of wound closure rates in cell-based treatments and topical therapies at various points compared with the untreated control wounds (P < .05). The results from the histologic, messenger RNA, and protein analyses suggested the treated wounds displayed increased angiogenesis and a diminished inflammatory response. ConclusionsCellular therapy with ASCs, EC/ASCs, and topical CM accelerated diabetic wound healing in the swine model. Enhanced angiogenesis and immunomodulation might be key contributors to this process.

A novel 3-dimensional (3D) microfluidics device to model the tumor microenvironment (TME).

704 Background: In vitro models of cancer have led to significant therapeutic advances. Despite the widespread use of in vitro tissue culture, the ability to directly evaluate human biology is limited by the inability to model the complex, 3D nature of the TME. We introduce a novel, microfluidic-based system of 3D human micro-tumors perfused with a network of human micro-vessels which could overcome the shortcomings of current in vitro systems. Methods: The micro-device was created by casting polydimethylsiloxane (PDMS) onto master molds, which are then bonded to a flat PDMS sheet using air plasma. Normal human lung fibroblasts (NHLF) and GFP labelled endothelial colony forming cell derived endothelial cells (EC-FCECs) were loaded in a fibrin gel at a 1:2 ratio into the central tissue chamber. Media was introduced through the microfluidic lines. The vascular network was developed with complete EGM2 media under nominal interstitial flow. Colorectal tumor cell lines labelled with mCherry were loaded to the side chambers on the seventh day after NHLF and EC loading. Bevacizumab or TGF- β were added on the second day after tumor cell loading. Results: Micro-vessels formed in the central chamber in 5-7 days after loading. The vessels were perfused with 70KDa fluorescent (red) dextran, and displayed intact vessel wall barrier. A suspension of a colorectal tumor cell line was loaded into the device side chambers, next to a fully developed vasculature. The tumor cells drove angiogenesis into the side chambers, and at the same time tumor began to migrate into the central chamber and within the vessel lumen. The angiogenesis induced by tumor cells can be pharmacologically inhibited, and the migration/ intravasation of tumor cells can be stimulated by TGF-β. Conclusions: Our novel micro-device system can be used as a functional in vitro system that can model the tumor micro-environment. This system has the advantage over current in vitro and in vivo systems in that it is high-throughput, rapid, cost-effective, and recreates many features of the 3D TME. We are currently expanding the platform to incorporate immune cells and designing a completely autologous system to test cancer immunotherapeutics.

Culturing with modified EGM2 medium enhances porcine neonatal islet-like cell clusters resistance to apoptosis in islet xenotransplantation.

Neonatal pig islet-like cell clusters (NICC) are an attractive source of insulin-producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood-mediated inflammatory reaction remains to be overcome. EGM2 medium depleted with hydrocortisone and supplemented with 50mmol/L isobutylmethylxanthine, 10mmol/L nicotinamide, and 10mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F-10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real-time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis-resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting. Compared with Ham's F-10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F-10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti-apoptotic Bcl-2 family member Mcl-1. Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation.

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial-like cells.

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD-MSCs) to characterize and differentiate them into endothelial-like cells. AD-MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony-forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM-2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial-like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD-MSCs showed their ability to form clones, to differentiate in vitro and the OCT-4, SOX-2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial-like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels-like structures. This study lays the foundations for future evaluation of the potential AD-MSCs pro-angiogenic and therapeutic role.

In vitro effects of propranolol and isoproterenol on the expression of beta-2 adrenergic receptor on infantile hemangioma endothelial cells

Objective To explore the role of beta-2 adrenergic receptor in the pathogenesis of infantile hemangioma. Methods In vitro cultured infantile hemangioma endothelial cells were divided into propranolol and isoproterenol groups. The propranolol groups were further classified into 5 groups to be treated with propranolol solutions at concentrations of 10, 15, 20 μg/ml, EGM-2 medium (blank control group 1) , and EGM-2 medium containing 0.16% DMSO (DMSO group) respectively, while the isoproterenol groups were classified into 4 groups to be treated with isoproterenol solutions at concentrations of 5, 10, 20 μg/ml and EGM-2 medium (blank control group 2) respectively. After 24-and 48-hour treatment, enzyme-linked immunosorbent assay (ELISA) was performed to measure the expression of beta-2 adrenergic receptor in cell culture supernatants in the above groups. Results After 24-hour treatment, 15-μg/ml and 20-μg/ml propranolol groups showed significantly decreased expression of beta-2 adrenergic receptor compared with blank control group 1 and DMSO group (all P < 0.05) . After 48-hour treatment, all the propranolol groups showed significantly decreased expression of beta-2 adrenergic receptor compared with the blank control group 1 (all P < 0.05) . However, the expression of beta-2 adrenergic receptor was significantly higher in the 10-and 20-μg/ml isoproterenol groups than in the blank control group 2 after 24-hour treatment (all P < 0.05) , and higher in the 20-μg/ml isoproterenol group than in the blank control group 2 after 48-hour treatment (P < 0.05) . Conclusion Propranolol can down-regulate the expression of beta-2 adrenergic receptor on the surface of vascular endothelial cells, while isoproterenol can up-regulate its expression. Key words: Hemangioma; Infant; Endothelial cells; Propranolol; Isoproterenol; Receptors, adre-nergic, beta-2

Abstract 166: Acceleration of Diabetic Wound Healing With Adipose Derived Stem Cells, Endothelial Differentiated Stem Cells and Topical Conditioned Medium Therapy in a Swine Model

Objectives: The goal of our study is to investigate the effect of adipose derived stem cells (ASCs), endothelial differentiated (ED) ASCs, and various conditioned medium (CM) on wound healing in a diabetic swine model. Methods: Diabetes was induced in 4 male Yorkshire pigs via intravenous injection of 150 mg/kg Streptozotocin. Pig ASCs were harvested and cultured in either M199 or EGM-2 medium with 30 mm glucose. Fourteen circular dorsal wounds (5 cm diameter; 5 mm depth) were created. Duplicate wounds were treated with one of 4 cell-based therapies by injection of 5 million (M) ASCs, 10M ASCs, 5M ED ASCs, or 10M ED ASCs on day 0 and half the initial dose on day 15. Wounds assigned to the topical CM therapy were covered with 2 cc of either serum free M199 primed by ASCs or Human Umbilical Vein Endothelial Cells every 3 days. One set of wound was designated as control. Wounds were assessed at day 0, 10, 15, 20 and 28. Animals were sacrificed on day 28. Image J software was used to evaluate percent wound healing. Results: On day 10, 15, 20 and 28 there was a significant increase in percent wound closure rates in both cell-based treatments when compared to control (p&lt;0.05, Figure 1 ). All treatments displayed significant increase in wound closure rates on day 28 when compared to control. Histology revealed a decrease in dermal inflammatory cells and improved restoration of normal skin architecture in therapeutic treatment groups when compared to untreated controls. Conclusions: Cellular therapy with ASCs, ED ASCs and topical CM accelerate diabetic wound healing in the swine model. Cellular therapy may provide the greatest benefit, as cells may continuously supply growth factors and adapt to the evolving microcellular environment as the wound progresses through the various phases of healing

Human Endothelial-Like Cells Transdifferentiated from CD14 + Monocytes Shows Suppressive Activity to Allo-Reactive T Cells

Although it is well known that liver allografts are often accepted by recipients, leading to donor-specific tolerance of further organ transplants, the underlying mechanisms remain unclear. We had previously showed that mouse liver sinusoidal endothelial cells (LSECs) had been proven capable of suppressing T-cells with cognate specificity for LSECs through programmed death (PD) 1/PD-L1 pathway in both of in vitro and in vivo models. Such immune-regulatory functions of LSECs have not been extensively investigated in human because of the limited availability of appropriate human samples. In this study, we have determined immune-regulatory properties of human LSECs on allogeneic T cells. We have also proven that endothelial-like cells, phenotypically and functionally resembling LSECs, can be transdifferentiated from human peripheral blood monocytes. Human LSECs were isolated from the disaggregated liver tissues obtained from patients undergoing surgical resection of colorectal liver metastasis. Human CD14+ cells were isolated from peripheral blood by using magnetic sorting and cultured in EGM2 medium supplemented with GM-CSF, IL-4, VEGF, hFGF-B, R3-IGF-1, and LPS for 14 days. Naïve human LSECs constitutively express molecules necessary for antigen presentation, i.e., freshly isolated CD105+ LSECs express MHC-class II, CD40, CD80, and CD86 together with PD-L1. The transdifferentiated cells morphologically appeared endothelial-like cells and phenotypically resembled LSECs, i.e., they were MHC-class II+, CD40low, CD80low, CD86low, CD105+, PDL-1+. The suppressive activity of those cells on allogeneic T cells was analyzed by a MLR assay using CFSE labeling technique. Allo-immune responses of both of CD4+ and CD8+ T cells in the MLR assays were significantly suppressed by subsequently adding the stimulator-type transdifferentiated endothelial-like cells, whereas there were not suppressed in response to anti-third party stimuli. The PD-1 expression on allo-reactive T cells among the final products of the MLR assays was elevated over time during culture. We have proven that human endothelial-like cells transdifferentiated from monocytes phenotypically resemble LSECs and suppress alloreactive T cells probably through PD-1/PD-L1 pathway. These findings raise a novel concept to induce hyporesponsiveness of alloreactive T cells by inoculating donor-derived those cells even in transplantation of organs other than liver.