Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.drug delivery | induced pluripotent stem cells | reprogramming | protein delivery | nanoparticle delivery I ntracellular delivery of macromolecules is a critical step in therapeutic and research applications. Nanoparticle-mediated delivery of DNA and RNA, for example, is being explored for gene therapy (1, 2), while protein delivery is a promising means of affecting cellular function in both clinical (3) and laboratory (4) settings. Other materials, such as small molecules, quantum dots, or gold nanoparticles, are of interest for cancer therapies (5, 6), intracellular labeling (7,8), and single-molecule tracking (9).The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles (10, 11), liposomes (12), or chemical modifications of the target molecule (13), such as cell-penetrating peptides (CPPs) (14, 15), to facilitate membrane poration or endocytotic delivery. In these methods, the delivery vehicle's efficacy is often dependent on the structure of the target molecule and the cell type. These methods are thus efficient in the delivery of structurally uniform materials, such as nucleic acids, but often ill-suited for the delivery of more structurally diverse materials, such as proteins (16,17) and some nanomaterials (7). Moreover, the endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles (18). More effective gene delivery methods, such as viral vectors (19,20), however, often risk chromosomal integration and are limited to DNA and RNA delivery.Membrane poration methods, such as electroporation (21, 22) and sonoporation (23), are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA (24) and ...

Mechanical forces are critical to embryogenesis, specifically, in the lineage-specification gastrulation phase, whereupon the embryo is transformed from a simple spherical ball of cells to a multi-layered organism, containing properly organized endoderm, mesoderm, and ectoderm germ layers. Several reports have proposed that such directed and coordinated movements of large cell collectives are driven by cellular responses to cell deformations and cell-generated forces. To better understand these environmental-induced cell changes, we have modeled the germ layer formation process by culturing human embryonic stem cells (hESCs) on three dimensional (3D) scaffolds with stiffness engineered to model that found in specific germ layers. We show that differentiation to each germ layer was promoted by a different stiffness threshold of the scaffolds, reminiscent of the forces exerted during the gastrulation process. The overall results suggest that three dimensional (3D) scaffolds can recapitulate the mechanical stimuli required for directing hESC differentiation and that these stimuli can play a significant role in determining hESC fate.

Successful tissue engineering requires optimization of scaffold stiffness for a given application and cell type. Here, we investigated the effect of scaffold stiffness on myoblast cells, demonstrating the ability of cells to affect and to sense their mechanical microenvironment. Myoblasts were cultured on composite three-dimensional poly-lactic acid (PLLA)/poly-lactic co glycolic acid (PLGA) porous scaffolds of varied elasticity. The elasticity was controlled by changing the ratio of PLLA versus PLGA in the scaffolds. Cell organization, myotube formation, and cell viability were affected by scaffold stiffness. PLLA-containing scaffolds (100% to 25% PLLA) provided stiffness that supported myotube formation, while neat PLGA scaffold failed to support myotube formation and cell viability. Furthermore, scaffold stiffness correlated to its size/area reduction upon culturing experiments, suggesting different shrinkage degree by cell forces. Inhibition of scaffold shrinking by affixing device resulted in spacious cell organization with normal cell morphology. This may suggest that scaffold shrinkage led to cellular degeneration and shape deformation. Our results indicate that compliant scaffolds are insufficient to withstand cell forces. On the other hand, excessively firm scaffold could not lead to parallel oriented myotube organization. Hence, optimal scaffold stiffness can be tailored by PLLA/PLGA blending to direct specific stages of myoblast differentiation and organization.

A hallmark of cancer cells is the metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon referred to as the ‘Warburg effect’, which is also observed in primed human pluripotent stem cells (hPSCs). Here, we report that downregulation of SIRT2 and upregulation of SIRT1 is a molecular signature of primed hPSCs and that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase. Remarkably, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased OXPHOS and increased glycolysis. In addition, we found that miR-200c-5p specifically targets SIRT2, downregulating its expression. Furthermore, SIRT2 overexpression in hPSCs significantly affected energy metabolism, altering stem cell functions such as pluripotent differentiation properties. Taken together, our results identify the miR-200c–SIRT2 axis as a key regulator of metabolic reprogramming (Warburg-like effect), via regulation of glycolytic enzymes, during human induced pluripotency and pluripotent stem cell function.

Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realized. Among these obstacles is the development of a productive method of isolating endothelial cells from human embryonic stem cells and elucidating their differentiation pathway. This review will focus on the endothelial potential of human embryonic stem cells that is de- Tissue vascularization and the clinical importance of endothelial progenitor cellsProgenitor endothelial cells (ECs) are promising key factors for many therapeutic applications. These applications include the following: cell transplantation for the repair of ischemic tissues, formation of blood vessels and heart valves, engineering of artificial vessels, repair of damaged vessels, and inducing the formation of blood vessel networks in engineered tissues. [1][2][3] Vascularization of engineered tissue in vitro before transplantation is essential for building complex and thick tissues because it enhances cell viability during tissue growth, induces structural organization, and promotes integration following implantation.Another area in which embryonic ECs can be beneficial is the study of human embryogenesis. In particular, ECs can serve as a model system for exploring central issues in human vasculogenesis and potentially elucidate vasculogenic and angiogenic mechanisms involved in the pathogenesis of vascular disease. Furthermore, it recently became evident that blood vessels do not just exchange metabolites between blood and tissue but play a more fundamental role in providing developmental cues to organs and differentiating cells. Early development of the pancreas depends on the presence of blood vessels, even in the absence of blood flow. [4][5][6] A similar dependence on ECs for the development of the liver and kidney has also been reported. 7,8 Angioblasts, or progenitor ECs, are associated with emerging buds of the embryonic lung and the nascent glandular portion of the stomach. 7 Studies of neuronal stem cell proliferation and differentiation lend further support to the view that organs must develop in proximity to blood vessels. 9 The potential overlap in signaling that occurs during neurogenesis and angiogenesis may even suggest that neurogenesis is regulated, in part, by an equilibrium between peripherally derived and centrally derived signaling molecules acting on both cell populations. Indeed, it has been shown that dividing cells in the mature hippocampus are immunoreactive for endothelial markers, thereby demonstrating the existence of neurogenesis within an angiogenic niche. 10 All these findings raise the possibility that endothelial signalin...