Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Cellular remodeling of fibrotic conduit as vascular graft.

The replacement of small-diameter arteries remains an unmet clinical need. Here we investigated the cellular remodeling of fibrotic conduits as vascular grafts. The formation of fibrotic conduit around subcutaneously implanted mandrels involved not only fibroblasts but also the trans-differentiation of inflammatory cells such as macrophages into fibroblastic cells, as shown by genetic lineage tracing. When fibrotic conduits were implanted as vascular grafts, the patency was low, and many fibrotic cells were found in neointima. Decellularization and anti-thrombogenic coating of fibrotic conduits produced highly patent autografts that remodeled into neoarteries, offering an effective approach to obtain autografts for clinical therapy. While autografts recruited mostly anti-inflammatory macrophages for constructive remodeling, allogenic DFCs had more T cells and pro-inflammatory macrophages and lower patency. Endothelial progenitors and endothelial migration were observed during endothelialization. Cell infiltration into DFCs was more efficient than decellularized arteries, and infiltrated cells remodeled the matrix and differentiated into smooth muscle cells (SMCs). This work provides insight into the remodeling of fibrotic conduits, autologous DFCs and allogenic DFCs, and will have broad impact on using fibrotic matrix for regenerative engineering.

Development of Injectable Amniotic Membrane Matrix for Postmyocardial Infarction Tissue Repair.

Ischemic heart disease represents the leading cause of death worldwide. Heart failure following myocardial infarction (MI) is associated with severe fibrosis formation and cardiac remodeling. Recently, injectable hydrogels have emerged as a promising approach to repair the infarcted heart and improve heart function through minimally invasive administration. Here, a novel injectable human amniotic membrane (hAM) matrix is developed to enhance cardiac regeneration following MI. Human amniotic membrane is isolated from human placenta and engineered to be a thermoresponsive, injectable gel around body temperature. Ultrasound-guided injection of hAM matrix into rat MI hearts significantly improves cardiac contractility, as measured by ejection fraction (EF), and decrease fibrosis. The results of this study demonstrate the feasibility of engineering as an injectable hAM matrix and its efficacy in attenuating degenerative changes in cardiac function following MI, which may have broad applications in tissue regeneration.

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's primary means of manufacturing therapeutic proteins, including monoclonal antibodies. The major challenge in cell line development for the production of recombinant biopharmaceuticals lies in generating and isolating rare high-producing stable clones, amongst thousands of low-producing or unstable clones, in a short period of time. One approach to accomplish this is to use the glutamine synthetase (GS) selection system, together with the GS inhibitor, methionine sulfoximine (MSX). However, MSX can only increase protein productivity to a limited extent. Often productivity will drop when MSX is removed from the system. We evaluated a congenital GS mutation, R324C, which causes glutamine deficiency in human as an attenuated selection marker for CHO cell line generation. We also created a panel of GS mutants with diminished GS activity. Our results demonstrated that using attenuated GS mutants as selection markers significantly increased antibody production of stably transfected pools. Furthermore, these stably transfected pools sustained high productivity levels for an extended period of time, whereas cells transfected with wild-type GS lost considerable protein productivity over time, particularly after MSX was removed. In summary, the use of attenuated GS as a selection marker in CHO cell line development bypasses the need for MSX, and generates stable clones with significantly higher antibody productivity.Abbreviations: CHO: Chinese hamster ovary; CMV: Cytomegalovirus; DHFR: Dihydrofolate reductase; GFP: Green fluorescent protein; GOI: gene-of-interest; GS: Glutamine synthetase; IRES: internal ribosomal entry site; MSX: Methionine sulfoximine; MTX: Methotrexate; psGS: pseudoGS; RVDs: Repeated variable di-residues; TALENs: transcription activator-like effector nucleases; VCD: Viable cell density; ZFNs: zinc finger nucleases.

Regeneration of a neoartery through a completely autologous acellular conduit in a minipig model: a pilot study.

BACKGROUND: Vascular grafts are widely used as a treatment in coronary artery bypass surgery, hemodialysis, peripheral arterial bypass and congenital heart disease. Various types of synthetic and natural materials were experimented to produce tissue engineering vascular grafts. In this study, we investigated in vivo tissue engineering technology in miniature pigs to prepare decellularized autologous extracellular matrix-based grafts that could be used as vascular grafts for small-diameter vascular bypass surgery. METHODS: Autologous tissue conduits (3.9 mm in diameter) were fabricated by embedding Teflon tubings in the subcutaneous pocket of female miniature pigs (n = 8, body weight 25-30 kg) for 4 weeks. They were then decellularized by CHAPS decellularization solution. Heparin was covalently-linked to decellularized tissue conduits by Sulfo-NHS/EDC. We implanted these decellularized, completely autologous extracellular matrix-based grafts into the carotid arteries of miniature pigs, then sacrificed the pigs at 1 or 2 months after implantation and evaluated the patency rate and explants histologically. RESULTS: After 1 month, the patency rate was 100% (5/5) while the inner diameter of the grafts was 3.43 ± 0.05 mm (n = 5). After 2 months, the patency rate was 67% (2/3) while the inner diameter of the grafts was 2.32 ± 0.14 mm (n = 3). Histological staining confirmed successful cell infiltration, and collagen and elastin deposition in 2-month samples. A monolayer of endothelial cells was observed along the inner lumen while smooth muscle cells were dominant in the graft wall. CONCLUSION: A completely autologous acellular conduit with excellent performance in mechanical properties can be remodeled into a neoartery in a minipig model. This proof-of-concept study in the large animal model is very encouraging and indicates that this is a highly feasible idea worthy of further study in non-human primates before clinical translation.

Biophysical regulation of cell reprogramming.

Induced pluripotent stem (iPS) cell reprogramming and direct reprogramming are promising approaches for disease modeling and personalized medicine. However, these processes are yet to be optimized. Biomaterials are increasingly integrated into cell reprogramming strategies in order to engineer the microenvironment, improve reprogramming efficiency and achieve effective in situ cell reprogramming. Although there are some studies on the role of biomaterials in iPS cell reprogramming, their effect on direct cell conversion has not been fully explored. Here we review the recent advances in the use of biomaterials for iPS cell reprogramming and direct reprogramming, with a focus on the biophysical aspect. We further highlight the future challenges and directions of the field.

3-Deazaneplanocin A and neplanocin A analogues and their effects on apoptotic cell death.

3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for the treatment of various cancers. DzNep has been reported to deplete histone methylations, including oncogenic EZH2 complex, giving rise to epigenetic modifications that reactivate many silenced tumor suppressors in cancer cells. Despite its promise as an anticancer drug, little is known about the structure-activity relationships of DzNep in the context of epigenetic modifications and apoptosis induction. In this study, a number of analogues of DzNep were examined for DzNep-like ability to induce synergistic apoptosis in cancer cells in combination with trichostatin A, a known histone deacetylase (HDAC) inhibitor. The structure-activity relationship data thus obtained provide valuable information on the structural requirements for biological activity. The studies identified three compounds that show similar activities to DzNep. Two of these compounds show good pharmacokinetics and safety profiles. Attempts to correlate the observed synergistic apoptotic activities with measured S-adenosylhomocysteine hydrolase (SAHH) inhibitory activities suggest that the apoptotic activity of DzNep might not be directly due to its inhibition of SAHH.

Cell-based proteome profiling using an affinity-based probe (AfBP) derived from 3-deazaneplanocin A (DzNep).

3-Deazaneplanocin A (DzNep), a global histone methylation inhibitor, has attracted significant interest in epigenetic research in recent years. The molecular mechanism of action and the cellular off-targets of DzNep, however, are still not well-understood. Our aim was to develop novel DzNep-derived small-molecule probes suitable to be used in live mammalian cells for identification of potential cellular targets of DzNep under physiologically relevant settings. In the current study, we have successfully designed, synthesized, and tested one such probe, called DZ-1. DZ-1 is a 'clickable' affinity-based probe (AfBP) derived from DzNep with minimal structural modifications. The probe was found to be highly cell-permeable, and possessed similar anti-apoptotic activities as DzNep in MCF-7 mammalian cells. Two additional control probes were made as negative labeling/pull-down probes in order to minimize false identification of background proteins due to unavoidable, intrinsic nonspecific photo-crosslinking reactions. All three probes were subsequently used for in-situ proteome profiling in live mammalian cells, followed by large-scale pull-down/LC-MS/MS analysis for identification of potential cellular protein targets that might interact with DzNep in native cellular environments. Our LC-MS/MS results revealed some highly enriched proteins that had not been reported as potential DzNep targets. These proteins might constitute unknown cellular off-targets of DzNep. Though further validation experiments are needed in order to unequivocally confirm these off-targets, our findings shed new light on the future use of DzNep as a validated chemical probe for epigenetic research and as a potential drug candidate for cancer therapy.