DNA methylation-mediated repression of exosomal miR-652-5p expression promotes oesophageal squamous cell carcinoma aggressiveness by targeting PARG and VEGF pathways.

Exosomal microRNAs (miRNAs) have been recently shown to play vital regulatory and communication roles in cancers. In this study, we showed that the expression levels of miR-652-5p in tumour tissues and serum samples of oesophageal squamous cell carcinoma (OSCC) patients were lower compared to non-tumorous tissues and serum samples from healthy subjects, respectively. Decreased expression of miR-652-5p was correlated with TNM stages, lymph node metastasis, and short overall survival (OS). More frequent CpG sites hypermethylation in the upstream of miR-652-5p was found in OSCC tissues compared to adjacent normal tissues. Subsequently, miR-652-5p downregulation promoted the proliferation and metastasis of OSCC, and regulated cell cycle both in cells and in vivo. The dual-luciferase reporter assay confirmed that poly (ADP-ribose) glycohydrolase (PARG) and vascular endothelial growth factor A (VEGFA) were the direct targets of miR-652-5p. Moreover, the delivery of miR-652-5p agomir suppressed tumour growth and metastasis, and inhibited the protein expressions of PARG and VEGFA in nude mice. Taken together, our findings provide novel insight into the molecular mechanism underlying OSCC pathogenesis.

Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when 'exosome-depleted serum' is utilised.

Extracellular vesicles (EVs) are important intercellular communication messengers. Half of the published studies in the field are in vitro cell culture based in which bovine serum in various concentrations and forms is used to facilitate the production of extracellular vesicles. 'Exosome depleted serum' is the type of bovine serum most widely used in the production of human EVs. Herein, we demonstrate that, despite the initial caution raised in 2014 about the persistence of bovine EVs, 'exosome depleted serum' was still used in 46% of publications on human or rodent EVs between 2015 and 2019. Using nanoparticle tracking analysis combined with detergent lysis of vesicles as well as bovine CD9 ELISA, we show that there were approximately 5.33 x 107/mL of bovine EVs remaining in the 'exosome depleted serum'. Importantly, the 'exosome depleted serum' was relatively enriched in small EVs by approximately 2.7-fold relative to the large EVs compared to that in the original serum. Specifically, the percentage of small EVs in total vesicles had increased from the original 48% in the serum before ultracentrifugation to 92% in the 'exosome depleted serum'. Furthermore, the pervasive bovine EVs carried over by the 'exosome depleted serum', even when the lowest concentration (0.5%) was used in cell culture, resulted in a significant contamination of human EVs in cell culture conditioned medium. Our findings indicate that the use 'exosome depleted serum' in cell culture-based studies may introduce artefacts into research examining the function of human and rodent EVs, in particular those involving EV miRNA. Thus, we appeal to the researchers in the EV field to seriously reconsider the practice of using 'exosome depleted serum' in the production of human and other mammalian EVs in vitro.

Serum or plasma? An old question looking for new answers.

Serum or plasma? An old question looking for new answers. There is a continual debate on what type of sample a clinical laboratory should use. While serum is still considered the gold standard and remains the required sample for some assays, laboratories must consider turn-around time, which is an important metric for laboratory performance and, more importantly, plays a critical role in patient care. In addition, a body of evidence emphasise the choice of plasma in order to prevent modifications of some analytes due to the coagulation process and related interferences. Advantages and disadvantages of serum and plasma are discussed on the basis of current literature and evidence. In addition, data are provided on the current utilisation of the samples (serum or plasma) in Italy and in other countries. Finally, a rationale for a possible switch from serum to plasma is provided.

Survival and immunomodulation of stem cells from human extracted deciduous teeth expanded in pooled human and foetal bovine sera.

The immunomodulatory properties of mesenchymal stem cells (MSCs) from autologous and allogeneic sources are useful in stimulating tissue regeneration and repair. To obtain a high number of MSCs for transplantation requires extensive in vitro expansion with culture media supplements that can cause xeno-contamination of cells potentially compromising function and clinical outcomes. In this study stem cells from human extracted deciduous teeth (SHED) were cultured in Knockout™ DMEM supplemented with either pooled human serum (pHS) or foetal bovine serum (FBS) to compare their suitability in maintaining immunomodulatory properties of cells during in vitro expansion. No significant difference in cell survival of SHED grown in pHS (pHS-SHED) or FBS (FBS-SHED) was observed when co-cultured with complement, monocytes or lymphocytes. However, significant changes in the expression of sixteen paracrine factors involved in immunomodulation were observed in the supernatants of FBS-SHED co-cultures with monocytes or lymphocytes compared to that in pHS-SHEDs after both 24 and 120 h of incubation. Further analysis of changing protein levels of paracrine factors in co-cultures using biological pathway analysis software predicted upregulation of functions associated with immunogenicity in FBS-SHED and lymphocyte co-cultures compared to pHS-SHED co-cultures. Pathway analysis also predicted significant stimulation of HMGB1 and TREM1 signalling pathways in FBS-SHED co-cultures indicating activation of immune cells and inflammation. Though FBS supplementation does not impact survival of SHED, our combinatorial biological pathway analysis supports the idea that in vitro expansion of SHEDs in pHS provides optimal conditions to minimise xeno-contamination and inflammation and maintain their immunomodulatory properties.

Antioxidant capacities in various animal sera as measured with multiple free-radical scavenging method.

Scavenging abilities of animal sera against six reactive species (OH, O2-, RO, t-BuOO, H3C, and 1O2) were determined with the use of multiple free-radical scavenging (MULTIS) method. Commercially available sera from pig, horse, rabbit, Guinea pig, hamster and chicken were subjected to MULTIS analysis and the results were compared with human specimen. In general, animal sera showed lower scavenging ability against OH and RO radicals than human serum. However, it is noteworthy that rabbit and chicken sera have higher scavenging ability against O2- than others. This is consistent with the known data that superoxide dismutase levels in these sera are high. In addition, we determined the uric acid level in animal sera using the uricase-TOOS method. In chicken serum, uric acid was found to be the major effective component in RO scavenging. This paper is first to quantitatively evaluate antioxidant capacities in animal sera.

Biobanking of different body fluids within the frame of IVF-a standard operating procedure to improve reproductive biology research.

PURPOSE: The aim of the present study was to develop a standard operating procedure (SOP) for the collection, transport, and storage of human cumulus cells, follicular fluid, blood serum, seminal plasma, embryo culture supernatant, and embryo culture supernatant control obtained within the IVF process under approved protocols and written informed consent from participating patients. The SOP was developed at the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz of the Medical University of Graz, Austria. METHODS: The SOP provides comprehensive details of laboratory procedures and sampling of the different fluids within the IVF process. Furthermore, information on sample coding, references of involved laboratory techniques (e.g., oocyte retrieval with a Steiner-TAN needle), ethical approvals, and biobanking procedures are presented. RESULTS: The result of the present study is a standard operating procedure. CONCLUSIONS: The SOP ensures a professional way for collection and scientific use of IVF samples by the Kinderwunsch Institut Schenk, Dobl, Austria, and Biobank Graz of the Medical University of Graz, Austria. It can be used as a template for other institutions to unify specimen collection procedures in the field of reproductive health research.

Elevated Levels of Total Cell-Free DNA in Maternal Serum Samples Arise from the Generation of Neutrophil Extracellular Traps.

INTRODUCTION: The analysis of cell-free DNA from maternal blood samples has facilitated the noninvasive detection of fetal aneuploidies or hereditary Mendelian disorders. In this context, previous studies have indicated that the pool of cell-free DNA is greater in maternal serum than in plasma samples, necessitating optimized collection and storage protocols. As the source of this increased amount of cell-free DNA is not clear, we have now examined whether neutrophil extracellular traps (NETs) contribute to this material. MATERIAL AND METHODS: Serum samples were collected in all three trimesters of normal healthy pregnant women, and at term from cases with manifest preeclampsia. The presence of NET-derived material was demonstrated by the detection of cell-free DNA fragments complexed to neutrophil granular proteins (i.e. myeloperoxidase). RESULTS: Our data indicate that NET-derived cell-free DNA/myeloperoxidase complexes were greater in serum from normal pregnant women than in normal matching nonpregnant controls. This neutrophil chromosomal material increased incrementally throughout gestation and was most pronounced in cases with preeclampsia. DISCUSSION: By detecting increased levels of cell-free DNA/myeloperoxidase complexes in maternal serum samples, our data indicate that a significant proportion of this material is derived from the generation of NETs.

Quantification of BCR-ABL mRNA in plasma/serum of patients with chronic myelogenous leukemia.

Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML) and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule) was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

[Changes of mitochondrial DNA/nuclear DNA ratio in the blood serum following X-ray irradiation of mice at various doses].

Using quantitative real-time PCR, the copy number of mitochondrial and nuclear DNA fragments in mouse blood serum was estimated at different time points following X-ray irradiation at various doses (from 0.5 to 10 Gy). The changes in the correlation between mtDNA and nuclear DNA (mtDNA/nucDNA) in blood serum reflect the degree of radiation injury depending on the dose of irradiation. Exposure to radiation at 10 Gy and massive cell death caused by this lethal dose result in a sharp decrease by an order of magnitude of the mtDNA/nucDNA ratio in the mouse serum; the value of this parameter did not recover within the next 3 days. The opposite effect was revealed when mice were exposed to irradiation at the dose of I Gy, which is not followed by massive cell death, but leads to a higher level of the mtDNA damage as compared with the nuclear DNA protected by histones. Defective mtDNA molecules enter the bloodstream, which results in an increase of the mtDNA/nucDNA ratio in serum. Under irradiation of mice at the intermediate dose of 3 Gy the two processes described above are exhibited at once. During the first hours after irradiation an apoptotic death of radiosensitive cells and release of a large number of nuclear DNA fragments in the serum are initiated, which reduces the mtDNA/nucDNA ratio. However, at later times after irradiation, starting from 5 days, an increase of the mtDNA/nucDNA ratio is observed in the serum, presumably as a result of reparation and elimination of defective mtDNA. Thus, the mtDNA/nucDNA ratio in the serum of irradiated mice reflects the degree of the radiation damage to cells and may be considered as a biological marker of radiation injury in the future.

Microfluidic device to study cell transmigration under physiological shear stress conditions.

The development of new drug therapies relies on studies of cell transmigration in in vitro systems. Migration has traditionally been studied using two methods, the Boyden chamber and a shear flow chamber assay. Though, commonly applied in cell transmigration studies, they are far from imitating a natural migration process. Here we describe a novel in vitro cell transmigration microfluidic assay, which mimicks physiological shear flow conditions in blood vessels. The device was designed to incorporate the principles of both the Boyden chamber and the shear flow chamber assay, i.e. migration through the membrane under flow conditions. The 3D environment of migrating cells is imitated by injecting cell adhesion proteins to coat the membrane in the device. We tested the developed device with Jurkat cells migration towards medium supplemented with serum, and with chemokine induced lymphocytes migration. The applied continuous flow of cell suspension and chemoattractant ensures that the concentration gradient is maintained in time and space. The cell adhesion proteins used to enhance cell migration in the device were fibronectin and VCAM-1. We successfully observed a multistep transmigration process by means of the developed microfluidic migration assay. The presented device is inexpensive, easy to fabricate and disposable, having a potential to be applied in basic research as well as in the drug development process.

The study of some haematological and serum biochemical parameters of juvenile beluga (Huso huso) fed oligofructose.

A study was conducted to investigate the effects of dietary oligofructose (1, 2 and 3%) on the blood profiles of beluga (Huso huso) juveniles (18.77 ± 0.76 g) compared to fish fed an un-supplemented diet. After 7 weeks of feeding on the experimental diets, haematological parameters, metabolic products (cholesterol, glucose and total protein) and serum enzymes (lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase) were measured. Compared to the control group (0% oligofructose), dietary oligofructose had no effect on red blood cell counts (RBC), mean corpuscular volume (MCV), mean cellular haemoglobin (MCH) or mean cell haemoglobin concentration (MCHC) (P > 0.05). However, haemoglobin (Hb) concentration, leucocyte (WBC) levels and the proportion of lymphocytes were significantly higher (P > 0.05) in the 2% oligofructose fed fish than in the 3% oligofructose fed fish. Additionally, haematocrit (Hct) values (P = 0.049) and the proportion of lymphocytes (P ≤ 0.01) were significantly higher in the 2% oligofructose group than in the control group. Although serum glucose and total protein remained unaffected, serum cholesterol was significantly lower in the 2% oligofructose group than in the control and 3% oligofructose group (P < 0.05). The results of the present study showed that oligofructose had no significant effects on serum lactate dehydrogenase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. These results indicate that fish blood profiles could be affected by prebiotics, which should be taken into account in future studies.

Non-contact microfluidic mechanical property measurements of single apoptotic bodies.

BACKGROUND: Cells exchange information by secreting micro- and nanosized extracellular vesicles (EVs), ranging from exosomes (30-100 nm) to apoptotic bodies (ABs, 1-5 µm). There is still much to understand about fundamental EV biological, physical, and chemical properties before clinical applications can be developed. EV mechanical properties have only been measured with atomic force microscopy (AFM) with its problematic adhesion and hard substrate effects. To understand EV mechanical behavior in less extreme mechanical conditions relevant to blood flow and many soft tissue environments, a non-contact measurement technique is needed. METHODS: We measured the mechanical properties of single microscale ABs derived from human blood plasma using non-contact microfluidics. EVs were gently stretched in extensional flow, similar to a traditional tensile test, and a linear mechanical model was applied to estimate mechanical stiffnesses from the observed stretching. RESULTS: The effective shear elastic modulus of ABs in non-contact flow conditions is approximately 5.6 ± 0.5 Pa, 7 orders of magnitude lower than previously reported AFM-measured biological exosome stiffnesses and 200 times smaller than suspended cells. CONCLUSIONS: Apoptotic bodies are very soft in fluid environments and exhibit lower effective stiffnesses than suspended cells. By measuring ABs in a natural fluid environment and low-force regime without hard probes and surfaces, we achieved closer agreement with linear mechanical theory and therefore more accurate stiffness measurements. GENERAL SIGNIFICANCE: AFM manufacturers and users should consider implementing new mechanical models to interpret AFM force indentation curves so that accurate extracellular vesicle mechanical properties can be extracted.

Microfluidic isolation and transcriptome analysis of serum microvesicles.

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

Autologous culture method improves retention of tumors' native properties.

No current in vitro tumor model replicates a tumor's in vivo microenvironment. A culturing technique that better preserves a tumor's pathophysiological conditions is needed for some important clinical applications, including personalized drug-sensitivity/resistance assays. In this study, we utilized autologous serum or body fluid to build a 3D scaffold and grow a patient's tumor. We named this technique "3D-ACM" (autologous culture method). Forty-five clinical samples from biopsies, surgically removed tumor tissues and malignant body fluids were cultured with 3D-ACM. Traditional 3D-FBS (fetal bovine serum) cultures were performed side-by-side for comparison. The results were that cells cultured in 3D-ACM rebuilt tissue-like structures, and retained their immuno-phenotypes and cytokine productions. In contrast, the 3D-FBS method promoted mesenchymal cell proliferation. In preliminary chemo drug-sensitivity assays, significantly higher mortality was always associated with FBS-cultured cells. Accordingly, 3D-ACM appears to more reliably preserve a tumor's biological characteristics, which might improve the accuracy of drug-testing for personalized cancer treatment.

Serum outperforms plasma in small extracellular vesicle microRNA biomarker studies of adenocarcinoma of the esophagus.

BACKGROUND: Circulating microRNAs (miRNAs) are potential biomarkers for many diseases. However, they can originate from non-disease specific sources, such as blood cells, and compromise the investigations for miRNA biomarkers. While small extracellular vesicles (sEVs) have been suggested to provide a purer source of circulating miRNAs for biomarkers discovery, the most suitable blood sample for sEV miRNA biomarker studies has not been defined. AIM: To compare the miRNA profiles between matched serum and plasma sEV preparations to determine their suitability for biomarker studies. METHODS: Matched serum and plasma samples were obtained from 10 healthy controls and 10 patients with esophageal adenocarcinoma. sEV isolates were prepared from serum and plasma using ExoQuickTM and quantified using NanoSight. RNA was extracted from sEV preparations with the miRNeasy Serum/Plasma kit and profiled using the Taqman Openarray qPCR. The overall miRNA content and the expression of specific miRNAs of reported vesicular and non-vesicular origins were compared between serum and plasma sEV preparations. The diagnostic performance of a previously identified multi-miRNA biomarker panel for esophageal adenocarcinoma was also compared. RESULTS: The overall miRNA content was higher in plasma sEV preparations (480 miRNAs) and contained 97.5% of the miRNAs found in the serum sEV preparations (412 miRNAs).The expression of commonly expressed miRNAs was highly correlated (Spearman's R = 0.87, P < 0.0001) between the plasma and serum sEV preparations, but was consistently higher in the plasma sEV preparations. Specific blood-cell miRNAs (hsa-miR-223-3p, hsa-miR-451a, miR-19b-3p, hsa-miR-17-5p, hsa-miR-30b-5p, hsa-miR-106a-5p, hsa-miR-150-5p and hsa-miR-92a-3p) were expressed at 2.7 to 9.6 fold higher levels in the plasma sEV preparations compared to serum sEV preparations (P < 0.05). In plasma sEV preparations, the percentage of protein-associated miRNAs expressed at relatively higher levels (Ct 20-25) was greater than serum sEV preparations (50% vs 31%). While the percentage of vesicle-associated miRNAs expressed at relatively higher levels was greater in the serum sEV preparations than plasma sEV preparations (70% vs 44%). A 5-miRNA biomarker panel produced a higher cross validated accuracy for discriminating patients with esophageal adenocarcinoma from healthy controls using serum sEV preparations compared with plasma sEV preparations (AUROC 0.80 vs 0.54, P < 0.05). CONCLUSION: Although plasma sEV preparations contained more miRNAs than serum sEV preparations, they also contained more miRNAs from non-vesicle origins. Serum appears to be more suitable than plasma for sEV miRNAs biomarkers studies.

[The specific features of the serum pattern in patients with pulmonary tuberculosis].

The specific features of the serum pattern were studied in 340 tuberculosis patients with different forms and phases of a specific process, by using wedge dehydration. In patients with tuberculosis, the predominant serum morphological type was established to be an ordered one with the radial pattern being preserved (50.3%) (r = 0.95); out of the pathological masses, toxic plaques (77.65%) and protein zone folds (66.2%) were prevalent (r = 0.98). In patients with circumscribe pulmonary tuberculosis and the productive course of the process, there was a preponderance of an ordered serum morphological type and indistinct markers of intoxication and lung tissue sclerosis. Disseminated tuberculosis with decay and complications was marked by a depressive serum morphological type [37.6% (r = 0.72)], severe metabolic disturbances, and markers of intoxication and lung tissue sclerosis.

Significant differences of function and expression of microRNAs between ground state and serum-cultured pluripotent stem cells.

Serum- and 2i-cultured embryonic stem cells (ESCs) show different epigenetic landscapes and transcriptomic profiles. The difference in the function and expression of microRNAs (miRNAs) between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with >100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8-/- or Dicer1-/- but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8-/- ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum- and 2i-cultured ESCs.

Functional assessment of the engraftment potential of gammaretrovirus-modified CD34+ cells, using a short serum-free transduction protocol.

The successful transduction and engraftment of human mobilized peripheral blood (MBP) CD34(+) cells are determined to a large extent by the ex vivo cell-processing conditions. In preparation for upcoming clinical trials, we investigated essential culture parameters and devised a short and efficient gammaretroviral transduction protocol entailing minimal manipulation of MBP CD34(+) cells. The engraftment potential and in vivo transgene expression in the progeny of repopulating CD34(+) cells were measured to assess the functionality of CD34(+) cells transduced under these conditions. Using a competitive in vivo repopulation assay in nonobese diabetic/severe combined immunodeficient mice, we demonstrate equivalent engraftment of CD34(+) cells transduced under serum-free conditions as compared with CD34(+) cells cultured with serum. We also took advantage of this in vivo model to demonstrate that ex vivo manipulation of CD34(+) cells can be shortened to 60 hr, using 36 hr of prestimulation and two cycles of transduction 12 hr apart. These minimally manipulated CD34(+) cells engraft in a manner similar to cells transduced under longer protocols and the vector-encoded transgene is expressed at the same frequency in cells derived from repopulating CD34(+) cells in vivo. We have thus developed a short and efficient human MBP CD34(+) transduction protocol under serum-free conditions that is suitable and broadly applicable for phase I clinical trials.

Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165.

Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.

Impact of serum source and inflammatory cytokines on the isolation of endothelial colony-forming cells from peripheral blood.

Endothelial colony-forming cells (ECFCs) isolated from peripheral blood are a highly promising cell source for a wide range of applications, including tissue engineering, in vivo vasculogenesis and anti-cancer therapeutics. Because of the potential for clinical translation, it is increasingly important to isolate and study ECFCs from patient cohorts that may benefit from such technologies. The primary objective of this investigation was to determine whether ECFCs could be obtained from patients with chronic kidney disease and diabetes (CKD-DM), using techniques that can be readily applied in the clinical setting. We also investigated the impact of autologous vs commercially available (i.e. allogeneic) human serum on ECFCs isolation. Surprisingly, the efficacy of ECFCs isolation from the CKD-DM group was comparable to a healthy control group when autologous serum was used. In contrast, substitution of allogeneic serum reduced ECFCs isolation in CKD-DM and control groups. In characterization studies, ECFCs were positive for several endothelial cell markers. ECFCs from the CKD-DM group were sensitive to inflammatory activation but their cellular proliferation was compromised. The concentrations of IL-4 and IL-8 were significantly increased in allogeneic serum, which induced a pro-inflammatory environment, including the release of IL-4, IL-6, IL-8 and MCP-1 into the conditioned media of cell cultures. Taken together, these data support further investigation into the use of autologous serum and cells for ECFC-based therapeutics and underscore the importance of the cytokine content in serum used for ECFCs isolation.