SummaryGene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm2. The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction.

Human cell-based 3D tissue constructs play an increasing role in disease modeling and drug screening. Inflammation, atherosclerosis, and many autoimmune disorders involve the interactions between immune cells and blood vessels. However, it has been difficult to image and model these interactions under realistic conditions. In this study, we fabricated a perfusion and imaging chamber to allow the real-time visualization of leukocyte perfusion, adhesion, and migration inside a tissue-engineered blood vessel (TEBV). We monitored the elevated monocyte adhesion to the TEBV wall and transendothelial migration (TEM) as the TEBV endothelium was activated by the inflammatory cytokine TNF-α. We demonstrated that treatment with anti-TNF-α or an NF-kB signaling pathway inhibitor would attenuate the endothelium activation and reduce the number of leukocyte adhesion (>74%) and TEM events (>87%) close to the control. As the first demonstration of real-time imaging of dynamic cellular events within a TEBV, this work paves the way for drug screening and disease modeling in TEBV-associated microphysiological systems.

Background: Human acellular dermal matrices (hADMs) are applied in various soft tissue reconstructive surgeries as scaffolds to support tissue remodeling and regeneration. To evaluate the clinical efficacy of hADM implants, it is integral that the hADM does not induce a host chronic inflammatory response leading to fibrotic encapsulation of the implant. In this study, we characterized the inflammatory and fibrosis-related tissue remodeling response of 2 commercial hADM products (SimpliDerm and AlloDerm RTU) in a nonhuman primate model using histology and gene expression profiling. Methods: Eighteen African green monkeys with abdominal wall defects were applied to evaluate the performance of SimpliDerm and AlloDerm RTU implants (N = 3) at 2, 4, and 12-weeks post-implantation. Using histology and gene expression profiling, tissue responses such as implant integration, degradation, cell infiltration, immune response, neovascularization, and pro-fibrotic responses over time were evaluated. Results: SimpliDerm showed a lower initial inflammatory response and slower implant degradation rate than AlloDerm RTU evidenced by histomorphological analysis. These factors led to a more anti-inflammatory and pro-remodeling microenvironment within SimpliDerm, demonstrated by lower TNFα levels and lower expression levels of pro-fibrotic markers, and promoted tissue repair and regeneration by 3-months post-implantation. Conclusions: Overall, histology and gene expression profiling analyses shown in this study demonstrated an effective model for analyzing hADM performance in terms of host inflammatory and fibrotic response. Further studies are warranted to fully evaluate the utility of this novel hADM in the clinical setting and verify the prognosis of our pre-clinical analysis model.

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.

Vascular smooth muscle cells (SMC) play an essential role in remodeling the vasculature during disease progression. Induced pluripotent stem cells (iPSC) provide an attractive approach to obtain autologous SMC source for patient-specific disease modeling. Here we discuss the current methods to 1) derive functional SMC from iPSC, 2) model vascular diseases using SMC generated from patient-derived iPSC, and 3) modulate microenvironmental cues to enhance cellular differentiation and functionality and better mimic the physiological environment. We emphasize that continuous exploration of biomaterial technologies to engineer a more SMC-specific microenvironment will provide further insight on complex vascular diseases.

Wound healing is a dynamic and complex process of replacing missing or dead cell structures and tissue layers. The aim of this research is to discover biocompatible materials and drugs that can promote cell migration in the wound area and thus enhance desirable wound healing effects. In this paper, we report that PDMS nanogratings could accelerate the migration of epithelial cells along the grating axis, and the addition of Imatinib could further increase the epithelial cell wound healing speed to 1.6 times the speed of control cells. We also demonstrate that this migration is mediated by lamellipodia protrusion, and is Rac1-GTPase activity dependent. Lastly, we discuss the potential application and prospect of different nanostructured biomaterials for wound healing studies.