l-Tryptophan-Induced Vasodilation Is Enhanced in Preeclampsia: Studies on Its Uptake and Metabolism in the Human Placenta.

l-tryptophan induces IDO (indoleamine 2,3-dioxygenase) 1-dependent vasodilation. IDO1 is expressed in placental endothelial cells and downregulated in preeclampsia. Hypothesizing that this may contribute to diminished placental perfusion, we studied l-tryptophan-induced vasodilation in healthy and early-onset preeclampsia placental arteries, focusing on placental kynurenine pathway alterations. Despite IDO1 downregulation, kynurenine pathway metabolite concentrations (measured with ultra-performance liquid chromatography-tandem mass spectrometry) were unaltered in preeclamptic versus healthy placentas. Most likely, this is due to enhanced l-tryptophan uptake, evidenced by increased l-tryptophan levels in preeclamptic placentas. Ex vivo perfused cotyledons from healthy and preeclamptic placentas released similar amounts of l-tryptophan and kynurenine pathway metabolites into the circulations. This release was not altered by adding l-tryptophan in the maternal circulation, suggesting that l-tryptophan metabolites act intracellularly. Maternally applied l-tryptophan did appear in the fetal circulation, confirming placental passage of this essential amino acid. After in vitro incubation of placental arteries with IDO1-upregulating cytokines interferon-γ and tumor necrosis factor-α, l-tryptophan induced vasodilation. This vasodilation was attenuated by both IDO1 and nitric oxide (NO) synthase inhibitors. Despite IDO1 downregulation, l-tryptophan-induced relaxation was enhanced in preeclamptic versus healthy placental arteries. However, cytokine stimulation additionally upregulated the LAT (l-type amino acid transporter) 1 in preeclamptic placental arteries only. Vasodilation to the lipophilic, transporter independent ethyl ester of l-tryptophan was reduced in preeclamptic versus healthy placental arteries, in agreement with reduced IDO1 expression. In conclusion, l-tryptophan induces IDO1- and NO-dependent relaxation in placental arteries, which is determined by l-tryptophan uptake rather than IDO1 expression. Increased l-tryptophan uptake might compensate for reduced IDO1 expression in preeclamptic placentas.

In Vivo Detection of Circulating Tumor Cells in High-Risk Non-Metastatic Prostate Cancer Patients Undergoing Radiotherapy.

High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique; further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%; p = 0.024) and yielded significantly higher CTC numbers (range: 0-15 vs. 0-5; p = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell's amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (<10 - >50%) and needs some further attention. This device is not cleared for the use in patients.

Endothelial indoleamine 2,3-dioxygenase-1 regulates the placental vascular tone and is deficient in intrauterine growth restriction and pre-eclampsia.

Indoleamine 2,3-dioxygenase-1 (IDO1) mediates the degradation of L-tryptophan (L-Trp) and is constitutively expressed in the chorionic vascular endothelium of the human placenta with highest levels in the microvasculature. Given that endothelial expression of IDO1 has been shown to regulate vascular tone and blood pressure in mice under the condition of systemic inflammation, we asked whether IDO1 is also involved in the regulation of placental blood flow and if yes, whether this function is potentially impaired in intrauterine growth restriction (IUGR) and pre-eclampsia (PE). In the large arteries of the chorionic plate L-Trp induced relaxation only after upregulation of IDO1 using interferon gamma and tumor necrosis factor alpha. However, ex vivo placental perfusion of pre-constricted cotyledonic vasculature with L-Trp decreases the vessel back pressure without prior IDO1 induction. Further to this finding, IDO1 protein expression and activity is reduced in IUGR and PE when compared to gestational age-matched control tissue. These data suggest that L-Trp catabolism plays a role in the regulation of placental vascular tone, a finding which is potentially linked to placental and fetal growth. In this context our data suggest that IDO1 deficiency is related to the pathogenesis of IUGR and PE.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

BACKGROUND: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Unravelling the biological secrets of microchimerism by single-cell analysis.

The presence of microchimeric cells is known for >100 years and well documented since decades. Earlier, microchimeric cells were mainly used for cell-based non-invasive prenatal diagnostics during early pregnancy. Microchimeric cells are also present beyond delivery and are associated to various autoimmune diseases, tissue repair, cancer and immune tolerance. All these findings were based on low complexity studies and occasionally accompanied by artefacts not allowing the biological functions of microchimerism to be determined. However, with the recent developments in single-cell analysis, new means to identify and characterize microchimeric cells are available. Cell labelling techniques in combination with single-cell analysis provide a new toolbox to decipher the biology of microchimeric cells at molecular and cellular level. In this review, we discuss how recent developments in single-cell analysis can be applied to determine the role and function of microchimeric cells.

Visualization of tumor heterogeneity by in situ padlock probe technology in colorectal cancer.

Tumor heterogeneity is considered a major cause for therapy resistance in colorectal cancer. Sub-populations of cells with different genetic alterations may exist in spatially distinct areas. Upon therapy, resistant sub-clones may enrich and ultimately lead to disease progression. Although ample data are available on tumors which are heterogeneous on a morphological level, only little is known about morphologically homogeneous tumors. We aimed to investigate if morphologically homogeneous colorectal cancer can harbor a heterogeneous genetic landscape. We chose to microdissect six morphologically homogeneous colorectal carcinomas into several areas and performed next-generation sequencing (NGS) to identify tumors with genetic heterogeneity. We then applied an mRNA-based in situ mutation detection technology based on padlock probes to localize and visualize mutations directly in the tumor tissue. In three out of six tumors, NGS revealed a high rate of variability of mutations between different tumor areas. We selected two cases for in situ mutation detection to visualize genetic heterogeneity. In situ mutation detection confirmed differences in mutant allele frequencies between different tumor areas of morphological homogeneous tumors. We conclude that genetic heterogeneity in morphologically homogeneous colorectal cancer is an observable, but underreported event. Our results illustrate the power of in situ mutation analysis to visualize genetic heterogeneity directly in tumor tissue.

Catch and Release: rare cell analysis from a functionalised medical wire.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single-cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Detection of Fetal Sex, Aneuploidy and a Microdeletion from Single Placental Syncytial Nuclear Aggregates.

OBJECTIVES: A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities. METHODS: SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH). RESULTS: DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy. CONCLUSION: SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities.

The role of placental tryptophan catabolism.

This review discusses the mechanisms and consequences of degradation of tryptophan (Trp) in the placenta, focusing mainly on the role of indoleamine 2,3-dioxygenase-1 (IDO1), one of three enzymes catalyzing the first step of the kynurenine pathway of Trp degradation. IDO1 has been implicated in regulation of feto-maternal tolerance in the mouse. Local depletion of Trp and/or the presence of metabolites of the kynurenine pathway mediate immunoregulation and exert antimicrobial functions. In addition to the decidual glandular epithelium, IDO1 is localized in the vascular endothelium of the villous chorion and also in the endothelium of spiral arteries of the decidua. Possible consequences of IDO1-mediated catabolism of Trp in the endothelium encompass antimicrobial activity and immunosuppression, as well as relaxation of the placental vasotonus, thereby contributing to placental perfusion and growth of both placenta and fetus. It remains to be evaluated whether other enzymes mediating Trp oxidation, such as indoleamine 2,3-dioxygenase-2, Trp 2,3-dioxygenase, and Trp hydroxylase-1 are of relevance to the biology of the placenta.

Are morphological criteria sufficient for the identification of circulating tumor cells in renal cancer?

BACKGROUND: Single circulating tumor cells (CTCs) or circulating tumor microemboli (CTMs) are potential biomarkers of renal cell cancer (RCC), however studies of CTCs/CTMs in RCC are limited. In this pilot study we aimed to evaluate a novel blood filtration technique suited for cytomorphological classification, immunocytochemical and molecular characterization of filtered, so called circulating non-hematologic cells (CNHCs) - putative CTCs/CTMs - in patients with RCC. METHODS: Blood of 40 patients with renal tumors was subjected to ScreenCell filtration. CNHCs were classified according to cytomorphological criteria. Immunocytochemical analysis was performed with antibodies against CD45, CD31 and carbonic anhydrase IX (CAIX, a RCC marker). DNA of selected CNHCs and respective primary tumors was analysed by array-CGH. RESULTS: CNHC-clusters with malignant or uncertain malignant cytomorphological features - putative CTMs - were negative for CD45, positive for CD31, while only 6% were CAIX positive. Array-CGH revealed that 83% of malignant and uncertain malignant cells did represent with a balanced genome whereas 17% presented genomic DNA imbalances which did not match the aberrations of the primary tumors. Putative single CTCs were negative for CD45, 33% were positive for CD31 and 56% were positive for CAIX. CONCLUSIONS: The majority of CNHC-clusters, putative CTMs, retrieved by ScreenCell filtration may be of endothelial origin. Morphological criteria seem to be insufficient to distinguish malignant from non-malignant cells in renal cancer.

Characterization of the conditioned medium from amniotic membrane cells: prostaglandins as key effectors of its immunomodulatory activity.

We previously demonstrated that cells isolated from the mesenchymal region of the human amniotic membrane (human amniotic mesenchymal tissue cells, hAMTC) possess immunoregulatory roles, such as inhibition of lymphocyte proliferation and cytokine production, and suppression of generation and maturation of monocyte-derived dendritic cells, as reported for MSC from other sources. The precise factors and mechanisms responsible for the immunoregulatory roles of hAMTC remain unknown. In this study, we aimed to identify the soluble factors released by hAMTC and responsible for the anti-proliferative effect on lymphocytes, and the mechanisms underlying their actions, in vitro. Conditioned medium (CM) was prepared under routine culture conditions from hAMTC (CM-hAMTC) and also from fragments of the whole human amniotic membrane (CM-hAM). We analyzed the thermostability, chemical nature, and the molecular weight of the factors likely responsible for the anti-proliferative effects. We also evaluated the participation of cytokines known to be involved in the immunomodulatory actions of MSC from other sources, and attempted to block different synthetic pathways. We demonstrate that the inhibitory factors are temperature-stable, have a small molecular weight, and are likely of a non-proteinaceous nature. Only inhibition of cyclooxygenase pathway partially reverted the anti-proliferative effect, suggesting prostaglandins as key effector molecules. Factors previously documented to take part in the inhibitory effects of MSCs from other sources (HGF, TGF-ß, NO and IDO) were not involved. Furthermore, we prove for the first time that the anti-proliferative effect is intrinsic to the amniotic membrane and cells derived thereof, since it is manifested in the absence of stimulating culture conditions, as opposed to MSC derived from the bone marrow, which possess an anti-proliferative ability only when cultured in the presence of activating stimuli. Finally, we show that the amniotic membrane could be an interesting source of soluble factors, without referring to extensive cell preparation.

Galectin-1 confers immune privilege to human trophoblast: implications in recurrent fetal loss.

Mechanisms accounting for the protection of the fetal semi-allograft from maternal immune cells remain incompletely understood. In previous studies, we showed that galectin-1 (Gal1), an immunoregulatory glycan-binding protein, hierarchically triggers a cascade of tolerogenic events at the mouse fetomaternal interface. Here, we show that Gal1 confers immune privilege to human trophoblast cells through the modulation of a number of regulatory mechanisms. Gal1 was mainly expressed in invasive extravillous trophoblast cells of human first trimester and term placenta in direct contact with maternal tissue. Expression of Gal1 by the human trophoblast cell line JEG-3 was primarily controlled by progesterone and pro-inflammatory cytokines and impaired T-cell responses by limiting T cell viability, suppressing the secretion of Th1-type cytokines and favoring the expansion of CD4(+)CD25(+)FoxP3(+) regulatory T (T(reg)) cells. Targeted inhibition of Gal1 expression through antibody (Ab)-mediated blockade, addition of the specific disaccharide lactose or retroviral-mediated siRNA strategies prevented these immunoregulatory effects. Consistent with a homeostatic role of endogenous Gal1, patients with recurrent pregnancy loss showed considerably lower levels of circulating Gal1 and had higher frequency of anti-Gal1 auto-Abs in their sera compared with fertile women. Thus, endogenous Gal1 confers immune privilege to human trophoblast cells by triggering a broad tolerogenic program with potential implications in threatened pregnancies.

Placental expression of indoleamine 2,3-dioxygenase.

This review focuses on the placental expression of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase-1 (IDO1) and its potential roles, which may not only encompass immunosuppression and antimicrobial activity, but also vasodilation based on the endothelial expression on both sides of the feto-maternal interface.

Exercise increases the frequency of circulating hematopoietic progenitor cells, but reduces hematopoietic colony-forming capacity.

Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2<0.15, equals 3,500 m, 3 h xposure) within a time span of at least 1 week. Blood was drawn from the cubital vein before and 10, 30, 60, and 120 min after exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (P<0.01). Most interestingly, although CD34+/CD45dim cells increased in number, the proliferative capacity of CPCs decreased significantly 10 min after cessation of exercise (P<0.05). A positive correlation between CPCs and MDA/MPO levels turned out to be significant for both normoxic and hypoxic conditions (P<0.05/P<0.01). Hypoxia did not provoke an additional effect. Although the CPC frequency increased, the functionality of CPCs decreased significantly after exercise, possibly due to the influence of increased oxidative stress levels.

Accession-specific haplotypes of the internal transcribed spacer region in Arabidopsis thaliana--a means for barcoding populations.

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.

Amnion-derived mesenchymal stromal cells show angiogenic properties but resist differentiation into mature endothelial cells.

Mesenchymal stromal cells derived from the human amnion (hAMSC) currently play an important role in stem cell research, as they are multipotent cells that can be isolated using noninvasive methods and are immunologically tolerated in vivo. The objective of this study was to evaluate their endothelial differentiation potential with regard to a possible therapeutic use in vascular diseases. hAMSC were isolated from human term placentas and cultured in Dulbecco's modified Eagle's medium (DMEM) (non-induced hAMSC) or endothelial growth medium (EGM-2) (induced hAMSC). Induced hAMSC changed their fibroblast-like toward an endothelial-like morphology, and were able to take up acetylated low-density lipoprotein and form endothelial-like networks in the Matrigel assay. However, they did not express the mature endothelial cell markers von Willebrand factor and vascular endothelial-cadherin. Gene expression analysis revealed that induced hAMSC significantly downregulated pro-angiogenic genes such as tenascin C, Tie-2, vascular endothelial growth factor A (VEGF-A), CD146, and fibroblast growth factor 2 (FGF-2), whereas they significantly upregulated anti-angiogenic genes such as serpinF1, sprouty1, and angioarrestin. Analysis of protein expression confirmed the downregulation of FGF-2 and Tie-2 (27%±8% and 13%±1% of non-induced cells, respectively) and upregulation of the anti-angiogenic protein endostatin (226%±4%). Conditioned media collected from hAMSC enhanced viability of endothelial cells and had a stabilizing effect on endothelial network formation as shown by lactate dehydrogenase and Matrigel assay, respectively. In summary, endothelial induced hAMSC acquired some angiogenic properties but resisted undergoing a complete differentiation into mature endothelial cells by upregulation of anti-angiogenic factors. Nevertheless, they had a survival-enhancing effect on endothelial cells that might be useful in a variety of cell therapy or tissue-engineering approaches.

Verification of the genomic identity of candidate microchimeric cells.

Microchimerism has been studied in the context of a variety of diseases which include autoimmune diseases (such as systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus and autoimmune thyroid diseases), cancer (e.g., of the cervix, thyroid gland, lung, breast), tissue repair, transplantation and transfusion. It may become relevant in the context of cell-based non-invasive prenatal diagnosis. But how to safely identify individual microchimeric cells? This is a nontrivial question, for which a solution has recently been suggested.