Direct Stem Cell Differentiation Research Articles

Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine.

Both lytic and temperate bacteriophages (phages) can be applied in nanomedicine, in particular, as nanoprobes for precise disease diagnosis and nanotherapeutics for targeted disease treatment. Since phages are bacteria-specific viruses, they do not naturally infect eukaryotic cells and are not toxic to them. They can be genetically engineered to target nanoparticles, cells, tissues, and organs, and can also be modified with functional abiotic nanomaterials for disease diagnosis and treatment. This Review will summarize the current use of phage structures in many aspects of precision nanomedicine, including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nanotherapeutics for targeted disease treatment. We will also propose future directions in the area of phage-based nanomedicines, and discuss the state of phage-based clinical trials.

Activation of Wnt/\u03b2-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

The generation of hepatocytes that are derived from human adipose stem cells (hASCs) represents an alternative to human hepatocytes for individualized therapeutic and pharmaceutical applications. However, the mechanisms facilitating hepatocyte differentiation from hASCs are not well understood. Here, we show that upon exposure to glycogen synthase kinase 3 (GSK3) inhibitors alone, the expression of definitive endoderm specific genes GATA4, FOXA2, and SOX17 in hASCs significantly increased in a manner with activation of Wnt/β-catenin signalling. Down regulation of the β-catenin expression attenuates the effect of GSK3 inhibitors on the induction of these specific genes. The cells induced using GSK3 inhibitors were directed to differentiate synchronously into hepatocyte-like cells (HLCs) after further combinations of soluble factors by a reproducible three-stage method. Moreover, hASC-HLCs induced using GSK3 inhibitors possess low-density lipoprotein uptake, albumin secretion, and glycogen synthesis ability, express important drug-metabolizing cytochrome P450 (CYP450) enzymes, and demonstrate CYP450 activity. Therefore, our findings suggest that activation of Wnt/β-catenin signalling via GSK3 inhibitors in definitive endoderm specification may represent an important mechanism mediating hASCs differentiated to functional hepatocyte. Furthermore, development of similar compounds may be useful for robust, potentially scalable and cost-effective generation of functional hepatocytes for drug screening and predictive toxicology platforms.

Long Noncoding RNAs in Pluripotency of Stem Cells and Cell Fate Specification.

Since the annotation of the mouse genome (FANTOM project) [Kawai J et al (2001) Functional annotation of a full-length mouse cDNA collection. Nature 409(6821):685-690] or the human genome [An integrated encyclopedia of DNA elements in the human genome. (2012) Nature 489(7414):57-74; Harrow J et al (2012) GENCODE: the reference human genome annotation for the ENCODE project. Genome Res 22(9):1760-1774], the roles of long noncoding RNAs in coordinating specific signaling pathways have been established in a wide variety of model systems. They have emerged as crucial and key regulators of stem cell maintenance and/or their differentiation into different lineages. In this chapter we have discussed the recently discovered lncRNAs that have been shown to be necessary for the maintenance of pluripotency of both mouse and human ES cells. We have also highlighted the different lncRNAs which are involved in directed differentiation of stem cells into any of the three germ layers. In recent years stem cell therapies including bone marrow transplantation are becoming an integral part of modern medicinal practices. However, there are still several challenges in making stem cell therapy more reproducible so that the success rate reaches a high percentage in the clinic. It is hoped that understanding the molecular mechanisms pertaining to the role of these newly discovered lncRNAs in the differentiation process of stem cells to specific lineages should pave the way to make stem cell therapy and regenerative medicine as a normal clinical practice in the near future.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Studies of the Influence of Gold Nanoparticles on Characteristics of Mesenchymal Stem Cells

The aim of the present study is to determine what effect the different concentrations of 15 nm gold nanoparticles (AuNPs) will have on the immunophenotype, synthesis collagen type I, ability to direct differentiation and spectroscopic characteristics of bone marrow mesenchymal stem cells (MSCs). The AuNPs in concentrations of 1.5–9 μg/ml did not lead to changes in the level of expression of CD 45, CD 90, and CD 73. It should be noted that AuNPs in concentrations of 6 and 9 μg/ml led to a decrease in CD 44 cells by 6% and 9%, respectively. The content of CD 105 cells was reduced by 5% when AuNPs were applied at a concentration of 9 μg/ml. It was found that AuNPs in concentrations of 1.5–6 μg/ml are safe for MSCs, while the increase up to 9 μg/ml has a toxic effect, manifested by the reduction of synthesis collagen type I and ability of adipogenic differentiation. IR spectroscopy data have shown that the AuNPs at concentrations of 9 μg/ml under conditions of adipogenic differentiation to MSCs lead to the destruction processes in the cells. The obtained results are related to the field of applied nanotechnology, which extends to regenerative medicine, especially in development of bioimplantology.

GATA6 is essential for endoderm formation from human pluripotent stem cells.

ABSTRACTProtocols have been established that direct differentiation of human pluripotent stem cells into a variety of cell types, including the endoderm and its derivatives. This model of differentiation has been useful for investigating the molecular mechanisms that guide human developmental processes. Using a directed differentiation protocol combined with shRNA depletion we sought to understand the role of GATA6 in regulating the earliest switch from pluripotency to definitive endoderm. We reveal that GATA6 depletion during endoderm formation results in apoptosis of nascent endoderm cells, concomitant with a loss of endoderm gene expression. We show by chromatin immunoprecipitation followed by DNA sequencing that GATA6 directly binds to several genes encoding transcription factors that are necessary for endoderm differentiation. Our data support the view that GATA6 is a central regulator of the formation of human definitive endoderm from pluripotent stem cells by directly controlling endoderm gene expression.

Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells.

Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. The microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction.

Chemical group-dependent plasma polymerisation preferentially directs adipose stem cell differentiation towards osteogenic or chondrogenic lineages.

Human adipose derived stem cells (hADSCs) are an exciting stem cell source for regenerative medicine due to their plentiful supply and ease of isolation. However, the optimal environmental cues to direct stem cells towards certain lineages change have to has not been identified. We have shown that by modifying the surface of the scaffold with specific chemical groups using plasma surface polymerisation techniques we can control ADSCs differentiation. This study shows that ADSCs can be differentiated towards osteogenic and chondrogenic lineages on amine (NH2) and carboxyl (COOH) modified scaffolds respectively. Plasma polymerisation can be easily applied to other biomaterial surfaces to direct stem cell differentiation for the regeneration of bone and cartilage.

Nanostructured scaffold as a determinant of stem cell fate.

The functionality of stem cells is tightly regulated by cues from the niche, comprising both intrinsic and extrinsic cell signals. Besides chemical and growth factors, biophysical signals are important components of extrinsic signals that dictate the stem cell properties. The materials used in the fabrication of scaffolds provide the chemical cues whereas the shape of the scaffolds provides the biophysical cues. The effect of the chemical composition of the scaffolds on stem cell fate is well researched. Biophysical signals such as nanotopography, mechanical forces, stiffness of the matrix, and roughness of the biomaterial influence the fate of stem cells. However, not much is known about their role in signaling crosstalk, stem cell maintenance, and directed differentiation. Among the various techniques for scaffold design, nanotechnology has special significance. The role of nanoscale topography in scaffold design for the regulation of stem cell behavior has gained importance in regenerative medicine. Nanotechnology allows manipulation of highly advanced surfaces/scaffolds for optimal regulation of cellular behavior. Techniques such as electrospinning, soft lithography, microfluidics, carbon nanotubes, and nanostructured hydrogel are described in this review, along with their potential usage in regenerative medicine. We have also provided a brief insight into the potential signaling crosstalk that is triggered by nanomaterials that dictate a specific outcome of stem cells. This concise review compiles recent developments in nanoscale architecture and its importance in directing stem cell differentiation for prospective therapeutic applications.

Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

Effects of nanoporous anodic titanium oxide on human adipose derived stem cells.

The aim of current bone biomaterials research is to design implants that induce controlled, guided, successful, and rapid healing. Titanium implants are widely used in dental, orthopedic, and reconstructive surgery. A series of studies has indicated that cells can respond not only to the chemical properties of the biomaterial, but also, in particular, to the changes in surface topography. Nanoporous materials remain in focus of scientific queries due to their exclusive properties and broad applications. One such material is nanostructured titanium oxide with highly ordered, mutually perpendicular nanopores. Nanoporous anodic titanium dioxide (TiO2) films were fabricated by a three-step anodization process in propan-1,2,3-triol-based electrolyte containing fluoride ions. Adipose-derived stem cells offer many interesting opportunities for regenerative medicine. The important goal of tissue engineering is to direct stem cell differentiation into a desired cell lineage. The influence of nanoporous TiO2 with pore diameters of 80 and 108 nm on cell response, growth, viability, and ability to differentiate into osteoblastic lineage of human adipose-derived progenitors was explored. Cells were harvested from the subcutaneous abdominal fat tissue by a simple, minimally invasive, and inexpensive method. Our results indicate that anodic nanostructured TiO2 is a safe and nontoxic biomaterial. In vitro studies demonstrated that the nanotopography induced and enhanced osteodifferentiation of human adipose-derived stem cells from the abdominal subcutaneous fat tissue.

Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications.

Poly(ethylene glycol) Hydrogels with Tailorable Surface and Mechanical Properties for Tissue Engineering Applications.

Advanced cellular biomanufacturing requires the large-scale production of biocompatible materials that can be utilized in the study of cell-matrix interactions and directed stem cell differentiation as well as the generation of physiologically relevant tissues for therapeutic applications. Herein we describe the development of a hydrogel based platform with tailorable mechanical properties that supports the attachment and proliferation of both pluripotent and multipotent stem cells. The biomimetic hydrogel scaffold generated provides biocompatible compositions for generating various tissue-like elasticities for regenerative medicine applications and advanced biomanufacturing.

A Generalized Gene-Regulatory Network Model of Stem Cell Differentiation for Predicting Lineage Specifiers.

SummaryIdentification of cell-fate determinants for directing stem cell differentiation remains a challenge. Moreover, little is known about how cell-fate determinants are regulated in functionally important subnetworks in large gene-regulatory networks (i.e., GRN motifs). Here we propose a model of stem cell differentiation in which cell-fate determinants work synergistically to determine different cellular identities, and reside in a class of GRN motifs known as feedback loops. Based on this model, we develop a computational method that can systematically predict cell-fate determinants and their GRN motifs. The method was able to recapitulate experimentally validated cell-fate determinants, and validation of two predicted cell-fate determinants confirmed that overexpression of ESR1 and RUNX2 in mouse neural stem cells induces neuronal and astrocyte differentiation, respectively. Thus, the presented GRN-based model of stem cell differentiation and computational method can guide differentiation experiments in stem cell research and regenerative medicine.

Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions.

Migration-driven aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface direct differentiation toward a cardiomyogenic fate commitment

Dynamic behaviors of cell aggregates on a dendrimer surface were investigated to drive the directed differentiation of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic lineage. Cell aggregates on the polyamidoamine dendrimer surface with fifth-generation (G5) of dendron structure showed dynamic changes in morphology associated with repetitive stretching and contracting during migration. Spatial-temporal observations revealed cellular movement in single aggregates by their morphological change through stretching and contracting on the G5 surface, suggesting that the dynamic behavior of aggregate causes mixing of cells. However, aggregates without cell-substrate adhesions on the low-binding culture surface sustained their spherical morphology without cellular movement within a single aggregate. Furthermore, β-catenin was observed at nuclei in aggregates on the G5 surface, and expression of the cardiomyocyte marker cardiac Troponin T (cTnT) was detected. However, β-catenin localized to the nuclei only in the outer region of the aggregate on the low-binding culture surface, and cTnT expression was restricted at the exterior surface of the aggregates. These observations indicate that cell mixing within aggregates on the G5 surface induced the directed differentiation of hMSCs toward a cardiomyogenic lineage by nuclear translocation of β-catenin through dissociation of cell-cell adhesions. These results suggest that migration-driven aggregate behaviors on the dendrimer surface caused repeated morphological changes of aggregate through stretching and contracting, leading to the directed differentiation of hMSCs toward a cardiomyogenic fate commitment.

Demethylation of SFRP2 by histone demethylase KDM2A regulated osteo-/dentinogenic differentiation of stem cells of the apical papilla.

Dental mesenchymal stem cells (MSCs) are easily obtained; however, mechanisms underlying directed differentiation of these cells remains unclear. Wnt/β-catenin signalling is essential for mesenchymal cell commitment and differentiation, and Wnt inhibition is linked to stem cell maintenance and function. Secreted frizzled-related protein 2 (SFRP2) competes with the Frizzled receptor for direct binding to Wnt and blocks activation of Wnt signalling. Here, we used stem cells derived from apical papillae (SCAPs) to study the functions of SFRP2. SCAPs were isolated from apical papillae of immature third molars. The cells were analysed using alkaline phosphatase activity assays, Alizarin red staining and quantitative calcium measurements. In addition, we evaluated expression profile of genes associated with osteogenesis and dentinogenesis (osteo-/dentinogenesis), and conducted in vivo transplantation experiments to determine osteo-/dentinogenic differentiation potential of SCAPs. ChIP assays were used to detect histone methylation at the SFRP2 promoter. We found that SFRP2 enhanced osteo-/dentinogenic differentiation via Osterix, a key transcription factor in SCAPs. Furthermore, silencing SFRP2 induced SCAP cell death in osteogenic-inducing medium, indicating that SFRP2 is a key factor in maintaining SCAP survival following osteo-/dentinogenic commitment. Moreover, we found that silencing KDM2A, a histone demethylase and BCL6 co-repressor, de-repressed SFRP2 transcription by increasing histone H3K4 and H3K36 methylation at the SFRP2 promoter. Our results have identified a new function of SFRP2 and shed new light on the molecular mechanism underlying directed differentiation of stem cells of dental origin.

Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons.

NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39–57), but not a Src-interfering peptide, Src(40–58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.

2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size

SummaryVariation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.

Hyaluronan Is Crucial for Stem Cell Differentiation into Smooth Muscle Lineage.

Deciphering the extracellular signals that regulate SMC differentiation from stem cells is vital to further our understanding of the pathogenesis of vascular disease and for development of cell‐based therapies and tissue engineering. Hyaluronan (HA) has emerged as an important component of the stem cell niche, however its role during stem cell differentiation is a complicated and inadequately defined process. This study aimed to investigate the role of HA in embryonic stem cell (ESC) differentiation toward a SMC lineage. ESCs were seeded on collagen‐IV in differentiation medium to generate ESC‐derived SMCs (esSMCs). Differentiation coincided with increased HA synthase (HAS) 2 expression, accumulation of extracellular HA and its assembly into pericellular matrices. Inhibition of HA synthesis by 4‐methylumbelliferone (4MU), removal of the HA coat by hyaluronidase (HYAL) or HAS2 knockdown led to abrogation of SMC gene expression. HA activates ERK1/2 and suppresses EGFR signaling pathways via its principle receptor, CD44. EGFR inactivation coincided with increased binding to CD44, which was further augmented by addition of high molecular weight (HMW)‐HA either exogenously or via HAS2 overexpression through adenoviral gene transfer. HMW‐HA‐stimulated esSMCs displayed a functional role in vascular tissue engineering ex vivo, vasculogenesis in a matrigel plug model and SMC accumulation in neointimal lesions of vein grafts in mice. These findings demonstrate that HAS2‐induced HA synthesis and organization drives ESC‐SMC differentiation. Thus, remodeling of the HA microenvironment is a critical step in directing stem cell differentiation toward a vascular lineage, highlighting HA as a potential target for treatment of vascular diseases. Stem Cells 2016;34:1225–1238