Efficient AAV9 Purification Using a Single-Step AAV9 Magnetic Affinity Beads Isolation.

Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results.

Wireless sequential dual light delivery for programmed PDT in vivo.

Using photodynamic therapy (PDT) to treat deep-seated cancers is limited due to inefficient delivery of photosensitizers and low tissue penetration of light. Polymeric nanocarriers are widely used for photosensitizer delivery, while the self-quenching of the encapsulated photosensitizers would impair the PDT efficacy. Furthermore, the generated short-lived reactive oxygen spieces (ROS) can hardly diffuse out of nanocarriers, resulting in low PDT efficacy.

Charting the next century of insulin replacement with cell and gene therapies.

The discovery of insulin a century ago changed the lives of millions of individuals suffering from diabetes, paving the way for long-term survival. While the availability of recombinant insulin for hormone replacement therapy has served extremely well to help control blood glucose in diabetes, there remains significant room for further improvements for an ultimate "cure" for diabetes patients. In this review, we celebrate the 100th anniversary of the discovery of insulin and consolidate the key milestones and advances in the development of recombinant human insulin.

First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model.

The only treatment tested for growth hormone receptor (GHR) defective Laron Syndrome (LS) is injections of recombinant insulin-like-growth factor 1 (rhIGF1). The response is suboptimal and associated with progressive obesity. In this study, we treated 4-5-week-old Laron dwarf mice (GHR-/-) with an adeno-associated virus expressing murine GHR (AAV-GHR) injection at a dose of 4 × 10 vector genome per mouse.

Identification of CD137-Expressing B Cells in Multiple Sclerosis Which Secrete IL-6 Upon Engagement by CD137 Ligand.

The potent costimulatory effect of CD137 has been implicated in several murine autoimmune disease models. CD137 costimulates and polarizes antigen-specific T cells toward a potent Th1/Tc1 response, and is essential for the development of experimental autoimmune encephalomyelitis (EAE), a murine model of Multiple Sclerosis (MS). This study aimed to investigate a role of CD137 in MS.

Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice.

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3' albumin enhancer (3'iALB) on insulin expression.

Angiopoietin-1 enhanced myocyte mitosis, engraftment, and the reparability of hiPSC-CMs for treatment of myocardial infarction.

Aims: To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure.

Methods And Results: Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro.

Tissues derived from reprogrammed Wharton's jelly stem cells of the umbilical cord as a platform to study gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) has been strongly associated with an increased risk of type 2 diabetes mellitus (T2DM) in later child and adulthood. The human umbilical cord and its contents are of fetal origin and represent the fetus genetically and physiologically. Since it is not possible to obtain tissues from the fetus and newborn to investigate the association between GDM and later T2DM, we reprogrammed the stem cells from the Wharton's jelly of umbilical cords (hWJSCs) of GDM and non-GDM mothers into induced pluripotent stem cells (iPSCs) and then differentiated the iPSCs into insulin-producing cells (IPCs) to provide pancreatic tissues that represent the fetus of GDM and normal mothers.

Fabrication of vascularized tissue constructs under chemically defined culture conditions.

Three-dimensional (3D) biofabrication techniques that enable the production of multicellular tissue equivalents for applications in basic biology, drug screening and regenerative medicne. Fabrication of these tissue constructs with in-built microvasculature enables recapitulation of the biological environment of the native tissues. Here, we present the fabrication of 3D vascularized tissue constructs containing microvascular networks using human embryonic stem cell (hESC)-derived endothelial cells (ECs) and pericytes encapsulated within a fibrin-based matrix and cultured under chemically defined conditions.

Inhibition of growth of Asian keloid cells with human umbilical cord Wharton's jelly stem cell-conditioned medium.

Background: Keloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known.

Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes.

Background: Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy.

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice.

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7-10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NOD mice.

Promoter optimisation of lentiviral vectors for efficient insulin gene expression in canine mesenchymal stromal cells: potential surrogate beta cells.

Background: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy.

Correction of Murine Diabetic Hyperglycaemia With A Single Systemic Administration of An AAV2/8 Vector Containing A Novel Codon Optimized Human Insulin Gene.

We report the correction of hyperglycemia of STZ induced diabetic mice using one intravenous systemic administration of a single stranded serotype 8 pseudotyped adeno-associated virus (ssAAV2/8) vector encoding the human proinsulin gene under a constitutive liver specific promoter. In vivo dose titration experiments were carried out and we identified an optimal range that achieved maintenance of euglycaemia or a mild diabetic condition for at least 9 months and ongoing to beyond 1 year for some animals, accompanied by human C-peptide secretion and weight gain. Further DNA codon optimization of the insulin gene construct resulted in approximately 3-10 times more human C-peptide secreted in the blood of codon optimized treated animals thereby reducing the number of vector particles required to achieve the same extent of reduction in blood glucose levels as the non-codon optimized vector.

Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies.

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes.

Grafts enriched with subamnion-cord-lining mesenchymal stem cell angiogenic spheroids induce post-ischemic myocardial revascularization and preserve cardiac function in failing rat hearts.

A crucial question in post-ischemic cell therapy refers to the ideal method of cell delivery to the heart. We hypothesized that epicardial implantation of subamnion-cord-lining mesenchymal stem cells (CL-MSC) angiogenic spheroids embedded within fibrin grafts (SASG) facilitates donor cell survival and enhances cardiac function in failing rat hearts. Furthermore, we compared the efficacy of this approach applied through two delivery methods.

Cord lining-mesenchymal stem cells graft supplemented with an omental flap induces myocardial revascularization and ameliorates cardiac dysfunction in a rat model of chronic ischemic heart failure.

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium.

Hydrogen sulfide suppresses outward rectifier potassium currents in human pluripotent stem cell-derived cardiomyocytes.

Aim: Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S.

Expression of CD137 on Hodgkin and Reed-Sternberg cells inhibits T-cell activation by eliminating CD137 ligand expression.

Hodgkin lymphoma is caused by a minority population of malignant Hodgkin and Reed-Sternberg (HRS) cells that recruit an abundance of inflammatory cells. The long-term survival of HRS cells among the vast majority of immune cells indicates that they have developed potent immune escape mechanisms. We report that the TNF receptor family member CD137 (TNFRSF9) is expressed on HRS cells, while normal B cells, from which HRS cells are most often derived, do not express CD137.

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells.

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming.

Post-ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid-enriched myocardial artificial grafts improves heart function in a rat model.

Angiogenesis plays a key role in post-ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)-enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP-luciferase-expressing rat cardiomyoblasts and enriched with 5 μm AA.

Keloid fibroblasts are more sensitive to Wnt3a treatment in terms of elevated cellular growth and fibronectin expression.

Background: Current evidence suggests the potential role of Wnt signalling in keloids pathogenesis but such literature remains scanty. We hypothesize that Wnt signalling is upregulated in keloid fibroblasts (KFs) and this promotes cellular growth, migration and extracellular matrix (ECM) production in such fibroblasts.

Objectives: To verify the downregulation of secreted frizzled-related protein 1 (SFRP1), a Wnt inhibitor and test KFs sensitivity to Wnt3a treatment compared to NFs in terms of activation of Wnt/β-catenin, cellular growth, migration and ECM expressions.

Characterization of human umbilical cord lining-derived epithelial cells and transplantation potential.

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines.

The role of R-spondin2 in keratinocyte proliferation and epidermal thickening in keloid scarring.

Keloids are found only in humans and the underlying biochemical mechanisms of their pathogenesis remain unknown. R-spondins (Rspos) are a relatively new group of secreted proteins known to be Wnt/β-catenin signaling agonists, but their role in keloids has yet to be elucidated. We investigated the expression levels of R-spondin2 (Rspo2) in cell lysates and conditioned media of monocultures and co-cultures of fibroblasts and keratinocytes derived from keloids and normal skin.