The self-renewal and differentiation of human pluripotent stem cells (hPSCs) have typically been studied in flat, two-dimensional (2D) environments. In this Perspective, we argue that 3D model ...systems may be needed in addition, as they mimic the natural 3D tissue organization more closely. We survey methods that have used 3D biomaterials for expansion of undifferentiated hPSCs, directed differentiation of hPSCs and transplantation of differentiated hPSCs in vivo.
Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not ...mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the self-renewal and differentiation of hESCs.
Currently, there are no clinically approved surgical glues that are nontoxic, bind strongly to tissue, and work well within wet and highly dynamic environments within the body. This is especially ...relevant to minimally invasive surgery that is increasingly performed to reduce postoperative complications, recovery times, and patient discomfort. We describe the engineering of a bioinspired elastic and biocompatible hydrophobic light-activated adhesive (HLAA) that achieves a strong level of adhesion to wet tissue and is not compromised by preexposure to blood. The HLAA provided an on-demand hemostatic seal, within seconds of light application, when applied to high-pressure large blood vessels and cardiac wall defects in pigs. HLAA-coated patches attached to the interventricular septum in a beating porcine heart and resisted supraphysiologic pressures by remaining attached for 24 hours, which is relevant to intracardiac interventions in humans. The HLAA could be used for many cardiovascular and surgical applications, with immediate application in repair of vascular defects and surgical hemostasis.
Here we report a two-step surface modification methodology to radiolabel small extracellular vesicles (SEVs) with 64CuCl2 for PET/MRI imaging. The modification did not change or damage the ...morphology, surface receptor proteins and internal RNA content. Radiolabeled SEVs could be detected in organs with low accumulation such as the brain (0.4-0.5% ID g-1) and their brain location determined by MRI.
Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Poly (lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates ...neovascularization and promotes wound healing. LL37 is an endogenous human host defense peptide that modulates wound healing and angiogenesis and fights infection. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) promotes wound closure due to the sustained release of both LL37 and lactate. In full thickness excisional wounds, the treatment with PLGA-LL37 NP significantly accelerated wound healing compared to PLGA or LL37 administration alone. PLGA-LL37 NP-treated wounds displayed advanced granulation tissue formation by significant higher collagen deposition, re-epithelialized and neovascularized composition. PLGA-LL37 NP improved angiogenesis, significantly up-regulated IL-6 and VEGFa expression, and modulated the inflammatory wound response. In vitro, PLGA-LL37 NP induced enhanced cell migration but had no effect on the metabolism and proliferation of keratinocytes. It displayed antimicrobial activity on Escherichia coli. In conclusion, we developed a biodegradable drug delivery system that accelerated healing processes due to the combined effects of lactate and LL37 released from the nanoparticles.
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The derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step ...differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 × 10−3 cm/min, a transendothelial electrical resistance value of 60 Ω cm2 and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells.
•Derivation of BCLECs from iPSCs in chemically defined medium in two steps•Specification of EPCs into BCLECs requires the activation of VEGF, Wnt3a, and RA•Fibronectin seems a sufficient substrate for the specification of EPCs into BCLECs
In this article, Ferreira and colleagues describe a protocol for the derivation of brain capillary-like endothelial cells from iPSCs under fully chemically defined medium and extracellular matrix in two steps. The protocol requires the modulation of three signaling pathways (VEGF, Wnt3a, and retinoic acid) and the use of fibronectin as a single extracellular matrix component.
We report that human embryonic stem cells contain a population of vascular progenitor cells that have the ability to differentiate into endothelial-like and smooth muscle (SM)-like cells. Vascular ...progenitor cells were isolated from EBs grown in suspension for 10 days and were characterized by expression of the endothelial/hematopoietic marker CD34 (CD34 cells). When these cells are subsequently cultured in EGM-2 (endothelial growth medium) supplemented with vascular endothelial growth factor-165 (50 ng/mL), they give rise to endothelial-like cells characterized by a cobblestone cell morphology, expression of endothelial markers (platelet endothelial cell-adhesion molecule-1, CD34, KDR/Flk-1, vascular endothelial cadherin, von Willebrand factor), incorporation of acetylated low-density lipoprotein, and formation of capillary-like structures when placed in Matrigel. In contrast, when CD34 cells are cultured in EGM-2 supplemented with platelet-derived growth factor-BB (50 ng/mL), they give rise to SM-like cells characterized by spindle-shape morphology, expression of SM cell markers (α-SM actin, SM myosin heavy chain, calponin, caldesmon, SM α-22), and the ability to contract and relax in response to common pharmacological agents such as carbachol and atropine but rarely form capillary-like structures when placed in Matrigel. Implantation studies in nude mice show that both cell types contribute to the formation of human microvasculature. Some microvessels contained mouse blood cells, which indicates functional integration with host vasculature. Therefore, the vascular progenitors isolated from human embryonic stem cells using methods established in the present study could provide a means to examine the mechanisms of endothelial and SM cell development, and they could also provide a potential source of cells for vascular tissue engineering.
Stimulation of adult neurogenesis by targeting the endogenous neural stem cells (NSCs), located in hippocampus and subventricular zone (SVZ), with nanoformulations has been proposed for brain repair ...in cases of neurodegenerative diseases. Unfortunately, it is relatively unknown the nanoformulation properties to facilitate their accumulation in the neurogenic niches after intravenous injection. Here, we have screened different gold-based formulations having variable morphology, surface chemistry and responsiveness to light for their capacity to cross the blood brain barrier (BBB) and accumulate preferentially in the neurogenic niches. Results obtained in a human in vitro BBB model showed that gold nanoparticles (Au NPs) and gold nanorods (Au NRs) conjugated with medium density of transferrin (Tf) peptides (i.e. between 169 and 230 peptides per NP) crossed more efficiently the BBB than the remaining formulations. This is due to a relatively lower avidity of these formulations to Tf receptor (TfR) and lower accumulation in the lysosomes, as compared to the other formulations. We further show that the near infrared light (NIR) irradiation of Au NRs, under a certain concentration and at specific cell culture time, lead to the opening of the BBB. Finally, we demonstrate that Au NRs conjugated with Tf administered intravenously in mice and activated by NIR had the highest accumulation in the neurogenic niches. Our results open the possibility of targeting more effectively the neurogenic niches by controlling the properties of the nanoformulations.
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Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human ...embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain >80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.
Abstract We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a ...dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF165 . The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including α -fetoprotein decreased 2- or 3-fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.
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