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Maternal depression and anxiety through pregnancy have lasting societal impacts. It is thus crucial to understand the trajectories of its progression from preconception to postnatal period, and the risk factors associated with it. Within the Bayesian framework, we propose to jointly model seven outcomes, of which two are physiological and five non-physiological indicators of maternal depression and anxiety over time. We model the former two by a Gaussian process and the latter by an autoregressive model, while imposing a multidimensional Dirichlet process prior on the subject-specific random effects to account for subject heterogeneity and induce clustering. The model allows for the inclusion of covariates through a regression term. Our findings reveal four distinct clusters of trajectories of the seven health outcomes, characterising women's mental health progression from before to after pregnancy. Importantly, our results caution against the loose use of hair corticosteroids as a biomarker, or even a causal factor, for pregnancy mental health progression. Additionally, the regression analysis reveals a range of preconception determinants and risk factors for depressive and anxiety symptoms during pregnancy.

This two-paper Series focuses on recent advances and applications of regenerative medicine that could benefit paediatric patients. Innovations in genomic, stem-cell, and tissue-based technologies have created progress in disease modelling and new therapies for congenital and incurable paediatric diseases. Prenatal approaches present unique opportunities associated with substantial biotechnical, medical, and ethical obstacles. Maternal plasma fetal DNA analysis is increasingly adopted as a noninvasive prenatal screening or diagnostic test for chromosomal and monogenic disorders. The molecular basis for cell-free DNA detection stimulated the development of circulating tumour DNA testing for adult cancers. In-utero stem-cell, gene, gene-modified cell (and to a lesser extent, tissue-based) therapies have shown early clinical promise in a wide range of paediatric disorders. Fetal cells for postnatal treatment and artificial placenta for ex-utero fetal therapies are new frontiers in this exciting field.

Aims To examine associations of assisted reproductive technology (ART) conception (vs. natural conception: NC) with offspring cardiometabolic health outcomes and whether these differ with age. Methods and results Differences in systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR), lipids, and hyperglycaemic/insulin resistance markers were examined using multiple linear regression models in 14 population-based birth cohorts in Europe, Australia, and Singapore, and results were combined using meta-analysis. Change in cardiometabolic outcomes from 2 to 26 years was examined using trajectory modelling of four cohorts with repeated measures. 35 938 (654 ART) offspring were included in the meta-analysis. Mean age ranged from 13 months to 27.4 years but was Conclusion These findings of small and statistically non-significant differences in offspring cardiometabolic outcomes should reassure people receiving ART. Longer-term follow-up is warranted to investigate changes over adulthood in the risks of hypertension, dyslipidaemia, and preclinical and clinical cardiovascular disease.

Asians are underrepresented across many omics databases, thereby limiting the potential of precision medicine in nearly 60% of the global population. As such, there is a pressing need for multi-omics derived quantitative trait loci (QTLs) to fill the knowledge gap of complex traits in populations of Asian ancestry. Here, we provide the first blood-based multi-omics analysis of Asian pregnant women, constituting high-resolution genotyping (N = 1079), DNA methylation (N = 915) and transcriptome profiling (N = 238). Integrative omics analysis identified 219 154 CpGs associated with cis-DNA methylation QTLs (meQTLs) and 3703 RNAs associated with cis-RNA expression QTLs (eQTLs). Ethnicity was the largest contributor of inter-individual variation across all omics datasets, with 2561 genes identified as hotspots of this variation; 395 of these hotspot genes also contained both ethnicity-specific eQTLs and meQTLs. Gene set enrichment analysis of these ethnicity QTL hotspots showed pathways involved in lipid metabolism, adaptive immune system and carbohydrate metabolism. Pathway validation by profiling the lipidome (~480 lipids) of antenatal plasma (N = 752) and placenta (N = 1042) in the same cohort showed significant lipid differences among Chinese, Malay and Indian women, validating ethnicity-QTL gene effects across different tissue types. To develop deeper insights into the complex traits and benefit future precision medicine research in Asian pregnant women, we developed iMOMdb, an open-access database.

Infant gastroesophageal reflux disease (GERD) is a significant cause of concern to parents. This study seeks to describe GERD prevalence in infants, evaluate possible risk factors and assess common beliefs influencing management of GERD among Asian parents.Mother-infant dyads in the Singapore PREconception Study of long-Term maternal and child Outcomes (S-PRESTO) cohort were prospectively followed from preconception to 12 months post-delivery. GERD diagnosis was ascertained through the revised Infant Gastroesophageal Reflux Questionnaire (I-GERQ-R) administered at 4 time points during infancy. Data on parental perceptions and lifestyle modifications were also collected.The prevalence of infant GERD peaked at 26.5% at age 6 weeks, decreasing to 1.1% by 12 months. Infants exclusively breastfed at 3 weeks of life had reduced odds of GERD by 1 year (adjusted odds ratio 0.43, 95% confidence interval 0.19-0.97,Prevalence of GERD in infants is highest in the first 3 months of life, and the majority outgrow it by 1 year of age. Infants exclusively breastfed at 3 weeks had reduced odds of GERD. Cultural-based changes such as elimination of "heaty or cold" food influence parental perceptions in GERD, which are unique to the Asian population. Understanding the cultural basis for parental perceptions and health-seeking behaviours is crucial in tailoring patient education appropriately for optimal management of infant GERD.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX (n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6–6.8% over 19–51 mo. The next cohort received 0.2–1 × 1013 vg boluses. AAV5-hFX animals (n = 3; 3.57 × 1013 vg/kg) expressed 35 mo. Low expressers (

Introduction: Superovulation-intrauterine insemination (SO-IUI) is the most common assisted reproductive technique (ART) in the world, with good evidence of efficacy and cost-effectiveness. However, parameters affecting its success have not been consistently reported. So in this study, we aim at determining the parameters influencing the success rate of SO-IUI. Materials and Methods: We conducted a retrospective cohort study of 797 SO-IUI cycles from 606 patients, performed between 2007 and 2009 in a single centre. These women received clomiphene citrate (CC), recombinant FSH (rFSH) or both. Results: There were 127 clinical pregnancies with a pregnancy rate (PR) of 15.9% (127/797) per treatment cycle. Factors associated with higher PR included maternal age

Mesenchymal stem cells (MSCs) possess some unique features (inherent tumor tropism, anti-inflammatory and immunosuppressive properties) that are not commonly found in conventional anti-cancer agents. These cells are known to secrete a vast array of proteins including growth factors, cytokines, chemokines, extracellular matrix metalloproteinases, and their corresponding inhibitors which exhibit profound effects on the microenvironment. However, the lack of a uniform method for culturing MSCs and their paracrine factors has hindered our understanding of MSC biology. In this chapter, we describe methods for the isolation, in vivo expansion, and phenotypic characterization of MSCs. In addition, methods for the collection and concentration of conditioned medium from these MSCs are described. Using tumor cells that constitutively express fluorescence reporter proteins, the effect of conditioned medium on tumor cell viability can be easily tested in vitro.

Application of dynamic mechanical loads on bone and bone explants has been reported to enhance osteogenesis and mineralization. To date, published studies have incorporated a range of cyclic strains on 3D scaffolds and platforms to demonstrate the effect of mechanical loading on osteogenesis. However, most of the loading parameters used in these studies do not emulate the in vivo loading conditions. In addition, the scaffolds/platforms are not representative of the native osteoinductive environment of bone tissue and hence may not be entirely accurate to study the in vivo mechanical loading. We hypothesized that biomimicry of physiological loading will potentiate accelerated osteogenesis in bone grafts. In this study, we present a compression bioreactor system that applies cyclic compression to cellular grafts in a controlled manner. Polycaprolactone-β Tricalcium Phosphate (PCL-TCP) scaffolds seeded with Mesenchymal Stem Cells (MSC) were cyclically compressed in bioreactor for a period of 4 weeks at 1 Hz and physiological strain value of 0.22% for 4 h per day. Gene expression studies revealed increased expressions of osteogenesis-related genes (Osteonectin and COL1A1) on day 7 of cyclic loading group relative to its static controls. Cyclic compression resulted in a 3.76-fold increase in the activity of Alkaline Phosphatase (ALP) on day 14 when compared to its static group (p 0.001). In addition, calcium deposition of cyclic loading group was found to attain saturation on day 14 (1.96 fold higher than its static scaffolds). The results suggested that cyclic, physiological compression of stem cell-seeded scaffolds generated highly mineralized bone grafts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2366-2375, 2017.

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.—Mattar, C. N., Wong, A. M. S., Hoefer, K., Alonso-Ferrero, M. E., Buckley, S. M. K., Howe, S. J., Cooper, J. D., Waddington, S. N., Chan, J. K. Y., Rahim, A. A. Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

STUDY QUESTION: Is implantation failure following ART associated with a perturbed decidual response in endometrial stromal cells (EnSCs)? SUMMARY ANSWER: Dynamic changes in the secretome of decidualizing EnSCs underpin the transition of a hostile to a supportive endometrial microenvironment for embryo implantation; perturbation in this transitional pathway prior to ART is associated with implantation failure. WHAT IS KNOWN ALREADY: Implantation is the rate-limiting step in ART, although the contribution of an aberrant endometrial microenvironment in IVF failure remains ill defined. STUDY DESIGN, SIZE, DURATION: In vitro characterization of the temporal changes in the decidual response of primary EnSCs isolated prior to a successful or failed ART cycle. An analysis of embryo responses to secreted cues from undifferentiated and decidualizing EnSCs was performed. The primary clinical outcome of the study was a positive urinary pregnancy test 14 days after embryo transfer. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary EnSCs were isolated from endometrial biopsies obtained prior to IVF treatment and cryopreserved. EnSCs from 10 pregnant and 10 non-pregnant patients were then thawed, expanded in culture, subjected to clonogenic assays, and decidualized for either 2 or 8 days. Transcript levels of decidual marker gene [prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1) and 11β-hydroxysteroid dehydrogenase (HSD11B1)] were analysed using real-time quantitative PCR and temporal secretome changes of 45 cytokines, chemokines and growth factors were measured by multiplex suspension bead immunoassay. The impact of the EnSC secretome on human blastocyst development was scored morphologically; and embryo secretions in response to EnSC cues analyzed by multiplex suspension bead immunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: Clonogenicity and induction of decidual marker genes were comparable between EnSC cultures from pregnant and non-pregnant group groups (P > 0.05). Analysis of 23 secreted factors revealed that successful implantation was associated with co-ordinated secretome changes in decidualizing EnSCs, which were most pronounced on Day 2 of differentiation: 17 differentially secreted proteins on Day 2 of decidualization relative to undifferentiated (Day 0) EnSCs (P < 0.05); 11 differentially secreted proteins on Day 8 relative to Day 2 (P < 0.05); and eight differentially secreted proteins on Day 8 relative to Day 0 (P < 0.05). By contrast, failed implantation was associated with a disordered secretome response. Blastocyst development was compromised when cultured for 24 h in medium conditioned by undifferentiated EnSCs when compared to decidualizing EnSCs. Analysis of the embryo microdroplets revealed that human blastocysts mount a secretory cytokine response to soluble decidual factors produced during the early (Day 2) but not late phase (Day 8) of differentiation. The embryo responses to secreted factors from decidualizing EnSCs were comparable between the pregnant and non-pregnant group (P > 0.05). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Although this study uses primary EnSCs and human embryos, caution is warranted when extrapolating the results to the in vivo situation because of the correlative nature of the study and limited sample size. WIDER IMPLICATIONS OF THE FINDINGS: Our finding raises the prospect that endometrial analysis prior to ART could minimize the risk of treatment failure. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by funds from the Biomedical Research Unit in Reproductive Health, a joint initiative of the University Hospitals Coventry & Warwickshire NHS Trust and Warwick Medical School, the University of Nottingham and Nurture Fertility, and the National Medical Research Council, Singapore (NMRC/BNIG14NOV023), the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, Flanders, Belgium), the "Fonds voor Wetenschappelijk Onderzoek" (FWO, Flanders, Belgium) and the "Wetenschappelijk Fonds Willy Gepts" (WFWG, UZ Brussel). The authors have declared that no conflict of interest exists.

To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity.We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application.Of 42 markers encompassing the α-globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co-amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop-out (ADO) rates of 0-5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls.This single-tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion-specific customization, and to linkage-based PGD of non-deletional Hb Bart's.

To develop an accurate non-invasive prenatal test using next generation sequencing (NGS) for HbE and the four most common β-thalassaemia mutations found in South East Asia (namely -28A G, CD17A T, CD41/42(-TTCT) and IVS-II-654C T).Cell-free DNA was extracted from maternal plasma from 83 families where both parents were carriers of the HbE mutation or one of four common β-thalassaemia mutations. Overlapping PCR amplicons covering each mutation were generated, pooled and sequenced using the Illumina MiSeq. Fastq files were analysed to detect inheritance of the paternal mutation.In two cases where the fathers were compound heterozygotes for HbE and -28A G, the fetus was correctly diagnosed as having inherited one of the paternal mutations. In 35/85 cases, the paternal mutation was not detected, and in 50/85 cases, it was classified as inherited. Overall sensitivity for detection of paternal mutations was 100% (95% CI: 92.4-100%), and specificity was 92.1% (95% CI: 79.2-97.3%).We demonstrated that detection of paternal mutations using NGS can be readily achieved with high sensitivity and specificity, removing the need for an invasive test in 50% of pregnancies at risk of a thalassaemia in cases where the father and mother carry a different mutation. © 2014 John WileySons, Ltd.

The objective of the study was to detect aneuploidy in single fetal nucleated red blood cells (FNRBCs) from placental villi using whole genome amplification (WGA) and next generation sequencing.Three single FNRBCs per sample were manually picked from villi collected from ten women undergoing elective first-trimester termination of pregnancy, and one or two cells were picked from each of four aneuploid chorionic villus samples. Following WGA and addition of adaptor and index sequences, samples were sequenced on the Illumina MiSeq. Leading and trailing 15 bases were trimmed, and reads were aligned to the human reference genome. Z-scores were calculated to determine deviation of the mean of the test from reference samples, with a score of 3 used as the threshold for classification of a particular chromosome as trisomic.We successfully made correct diagnoses from ten single cells isolated from villi from two cases of trisomy 21 (one case from a single cell and one from two cells), two cases of trisomy 18 (two cells each), and a case of trisomy 15 (three cells).With their faithful representation of fetal genome, diagnosis using single FNRBCs provides a definitive result compared with non-invasive prenatal testing using cell-free fetal DNA, and is a safer alternative to invasive amniocentesis.

Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a polysaccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force microscopy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modulus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline acetyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during electrospinning and suggests the feasibility of using such polysaccharide nanofibers in supporting fMSC neuronal commitment.

Geometric cues have been used for a variety of cell regulation and tissue regenerative applications. While the function of geometric cues is being recognized, their stability and degradation behaviors are not well known. Here, we studied the influence of degradation on uniaxial-stretch-induced poly(ε-caprolactone) (UX-PCL) ridge/groove arrays and further cellular responses. Results from accelerated hydrolysis in vitro showed that UX-PCL ridge/groove arrays followed a surface-controlled erosion, with an overall geometry remained even at ∼45% film weight loss. Compared to unstretched PCL flat surfaces and/or ridge/groove arrays, UX-PCL ridge/groove arrays achieved an enhanced morphological stability against degradation. Over the degradation period, UX-PCL ridge/groove arrays exhibited an "S-shape" behavior of film weight loss, and retained more stable surface hydrophilicity and higher film mechanical properties than those of unstretched PCL surfaces. Human mesenchymal stem cells (MSCs) aligned better toward UX-PCL ridge/groove arrays when the geometries were remained intact, and became sensitive with gradually declined nucleus alignment and elongation to the geometric degradation of ridges. We speculate that uniaxial stretching confers UX-PCL ridge/groove arrays with enhanced stability against degradation in erosive environment. This study provides insights of how degradation influences geometric cues and further cell responses, and has implications for the design of biomaterials with stability-enhanced geometric cues for long-term tissue regeneration.

Human mesenchymal stem cells (MSCs) derived from various origins show varied differentiation capability. Recent work shows that cell shape manipulation via micropatterning can modulate the differentiation of bone-marrow-derived MSCs. Herein, the effect of micropatterning on the myogenesis of MSCs isolated from three different sources (bone marrow, fetal tissue, and adipose) is reported. All the well-aligned cells, regardless of source, predominantly commit to myogenic lineage, as shown by the significant upregulation of myogenic gene markers and positive myosin heavy chain staining. It is demonstrated that our novel micropattern can be used as a generic platform for inducing myogenesis of MSCs from different sources and may also have the potential to be extended to induce other lineage commitment.

Intrauterine gene therapy (IUGT) potentially enables the treatment and possible cure of monogenic -diseases that cause severe fetal damage. The main benefits of this approach will be the ability to correct the disorder before the onset of irreversible pathology and inducing central immune tolerance to the vector and transgene if treatment is instituted in early gestation. Cure has been demonstrated in small animal models, but because of the significant differences in immune ontogeny and the much shorter gestation compared to humans, it is unlikely that questions of long-term efficacy and safety will be adequately addressed in rodents. The nonhuman primate (NHP) allows investigation of key issues, in particular, the different outcomes in early and late-gestation IUGT associated with different stages of immune maturity, longevity of transgene expression, and delayed-onset adverse events in treated offspring and mothers including insertional mutagenesis. Here, we describe a model based on the Macaca fascicularis using ultrasound and fetoscopic approaches to systemic vector delivery and the processes involved in vector administration and longitudinal analyses.

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings.

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.