Development and functional testing of a novel in vitro delayed scratch closure assay.

As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton's jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo.

An effective device to enable consistent scratches for in vitro scratch assays.

BACKGROUND: The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by mechanically removing cells through manual scraping with a sharp-edged tool. This step is traditionally done with pipette tips and is unsuitable for high-throughput assays, as the created scratches are highly variable in width and position. Commercially available solutions are often expensive, and require specific cultureware which might not be suitable for all studies. RESULTS: In this study, we have developed a flexible cell scratch device comprising a single wounding tool, a guide and an imaging template for consistent and reproducible scratch assays in 96-well plates. Our results showed that the device produced a more consistent scratch profile compared to the conventional method of using pipette tips. The imaging template also allowed operators to easily locate and image the same region of interest at different time points, which potentially could be used for other assays. CONCLUSIONS: Our flexible yet effective scratch device thus enables robust scratch assays that can be applied to different experimental needs, providing researchers with an easy and reliable tool for their studies.

Amelioration of X-Linked Related Autophagy Failure in Danon Disease With DNA Methylation Inhibitor.

BACKGROUND: Danon disease is an X-linked disorder that leads to fatal cardiomyopathy caused by a deficiency in lysosome-associated membrane protein-2 (LAMP2). In female patients, a later onset and less severe clinical phenotype have been attributed to the random inactivation of the X chromosome carrying the mutant diseased allele. We generated a patient-specific induced pluripotent stem cell (iPSCs)-based model of Danon disease to evaluate the therapeutic potential of Xi-chromosome reactivation using a DNA methylation inhibitor. METHODS: Using whole-exome sequencing, we identified a nonsense mutation (c.520C>T, exon 4) of the LAMP2 gene in a family with Danon disease. We generated iPSC lines from somatic cells derived from the affected mother and her 2 sons, and we then differentiated them into cardiomyocytes (iPSC-CMs) for modeling the histological and functional signatures, including autophagy failure of Danon disease. RESULTS: Our iPSC-CM platform provides evidence that random inactivation of the wild-type and mutant LAMP2 alleles on the X chromosome is responsible for the unusual phenotype in female patients with Danon disease. In vitro, iPSC-CMs from these patients reproduced the histological features and autophagy failure of Danon disease. Administration of the DNA demethylating agent 5-aza-2'-deoxycytidine reactivated the silent LAMP2 allele in iPSCs and iPSC-CMs in female patients with Danon disease and ameliorated their autophagy failure, supporting the application of a patient-specific iPSC platform for disease modeling and drug screening. CONCLUSIONS: Our iPSC-CM platform provides novel mechanistic and therapeutic insights into the contribution of random X chromosome inactivation to disease phenotype in X-linked Danon disease.

Pleiotrophin gene therapy for peripheral ischemia: evaluation of full-length and truncated gene variants.

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.

Donor myocardial infarction impairs the therapeutic potential of bone marrow cells by an interleukin-1-mediated inflammatory response.

Delivery of bone marrow cells (BMCs) to the heart has substantially improved cardiac function in most rodent models of myocardial infarction (MI), but clinical trials of BMC therapy have led to only modest improvements. Rodent models typically involve intramyocardial injection of BMCs from distinct donor individuals who are healthy. In contrast, autologous BMCs from individuals after MI are used for clinical trials. Using BMCs from donor mice after MI, we discovered that recent MI impaired BMC therapeutic efficacy. MI led to myocardial inflammation and an increased inflammatory state in the bone marrow, changing the BMC composition and reducing their efficacy. Injection of a general anti-inflammatory drug or a specific interleukin-1 inhibitor to donor mice after MI prevented this impairment. Our findings offer an explanation of why human trials have not matched the success of rodent experiments and suggest potential strategies to improve the success of clinical autologous BMC therapy.

Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines.

The role of selective nitric oxide synthase inhibitor on nitric oxide and PGE2 levels in refractory hemorrhagic-shocked rats.

BACKGROUND: The up-regulation of nitric oxide (NO) and cyclooxgenase-2 (COX-2) has been implicated in the pathophysiology of hemorrhagic shock. We examined the effects of aminoguanidine (AG), which is a known inducible nitric oxide synthase (iNOS) inhibitor, and NS-398, a known COX-2 inhibitor, in our rat model of refractory hemorrhagic shock (RHS). MATERIAL AND METHODS: We measured tissue iNOS and COX-2 protein expression, brain and plasma nitrate/nitrite and prostaglandin E2 (PGE2) levels, plasma creatinine and glutamic oxalacetic transaminase (GOT) levels, quantified the histological damages in kidney, liver, lung, and brain, survival rate, and mean arterial blood pressure (MABP) in RHS rats. RESULTS: Semiquantitative analysis of tissues showed iNOS protein was not detected in AG + RHS rats but was detected in normal saline and NS-398 RHS rats. Tissue COX-2 protein was not detected in AG and NS-398 RHS rats but was detected in normal saline + RHS rats. The levels of brain and plasma nitrate/nitrite and PGE2 and plasma creatinine and GOT were significantly lower in the AG + RHS rat group when compared with the normal saline RHS rat group. Histological examinations also showed a reduction in organ damage for AG + RHS rats when compared with treated RHS rats. AG + RHS rats showed significantly increased survival and MABP level when compared with treated RHS rats. CONCLUSION: Our present findings suggest that NO produced by iNOS might result in organ damages. This in turn might lead to COX-2 up-regulation, and it increases the production of reactive oxygen species and toxic prostanoids. NO-mediated organ damage might be one way in which toxic products of COX-2 might further contribute to NO's deleterious effect in the later stages of RHS. It is therefore suggested that treatment of AG via inhibition of NO might contribute to improved physiological parameters and survival rates following RHS.

Preservation of neurological functions by nitric oxide synthase inhibitors in conscious rats following delayed hemorrhagic shock.

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated in the pathophysiology of hemorrhagic shock. Our aim was to study the effects of NOS inhibitors, aminoguanidine (AG) and NG-nitro-L-arginine methyl ester (L-NAME), on survival rate, mean arterial blood pressure (MABP), temporal evolution of infarct volume, nitric oxide (NO) production and neurological deficit in a model of delayed hemorrhagic shock (DHS) in conscious rats. Our results showed that the NOS inhibitors significantly improved survival rate, MABP, and attenuated brain NO overproduction 24, 48 h and 72 h after DHS. AG reduced brain infarct volume and improved the neurological performance evaluated by the rotameric and grip strength tests while L-NAME did not show protective effect in rats following DHS. These findings suggest that NO formation via iNOS activation may contribute to organ damage and that the selective iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following DHS.