Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

Direct intrathecal implantation of mesenchymal stromal cells leads to enhanced neuroprotection via an NFkappaB-mediated increase in interleukin-6 production.

Mesenchymal stromal cell (MSC) therapy has shown promise for the treatment of traumatic brain injury (TBI). Although the mechanism(s) by which MSCs offer protection is unclear, initial in vivo work has suggested that modulation of the locoregional inflammatory response could explain the observed benefit. We hypothesize that the direct implantation of MSCs into the injured brain activates resident neuronal stem cell (NSC) niches altering the intracerebral milieu. To test our hypothesis, we conducted initial in vivo studies, followed by a sequence of in vitro studies. In vivo: Sprague-Dawley rats received a controlled cortical impact (CCI) injury with implantation of 1 million MSCs 6 h after injury. Brain tissue supernatant was harvested for analysis of the proinflammatory cytokine profile. In vitro: NSCs were transfected with a firefly luciferase reporter for NFkappaB and placed in contact culture and transwell culture. Additionally, multiplex, quantitative PCR, caspase 3, and EDU assays were completed to evaluate NSC cytokine production, apoptosis, and proliferation, respectively. In vivo: Brain supernatant analysis showed an increase in the proinflammatory cytokines IL-1alpha, IL-1beta, and IL-6. In vitro: NSC NFkappaB activity increased only when in contact culture with MSCs. When in contact with MSCs, NSCs show an increase in IL-6 production as well as a decrease in apoptosis. Direct implantation of MSCs enhances neuroprotection via activation of resident NSC NFkappaB activity (independent of PI3 kinase/AKT pathway) leading to an increase in IL-6 production and decrease in apoptosis. In addition, the observed NFkappaB activity depends on direct cell contact.

Modern approaches to pediatric brain injury therapy.

Each year, pediatric traumatic brain injury (TBI) accounts for 435,000 emergency department visits, 37,000 hospital admissions, and approximately 2,500 deaths in the United States. TBI results in immediate injury from direct mechanical force and shear. Secondary injury results from the release of biochemical or inflammatory factors that alter the loco-regional milieu in the acute, subacute, and delayed intervals after a mechanical insult. Preliminary preclinical and clinical research is underway to evaluate the benefit from progenitor cell therapeutics, hypertonic saline infusion, and controlled hypothermia. However, all phase III clinical trials investigating pharmacologic monotherapy for TBI have shown no benefit. A recent National Institutes of Health consensus statement recommends research into multimodality treatments for TBI. This article will review the complex pathophysiology of TBI as well as the possible therapeutic mechanisms of progenitor cell transplantation, hypertonic saline infusion, and controlled hypothermia for possible utilization in multimodality clinical trials.

A glance on recent progresses in diagnosis and treatment of primary immunodeficiencies

Primary immunodeficiencies (PIDs)* belong to the group of rare diseases which need more awareness by the relevant medical disciplines. Below a review on recent progresses in diagnosis and treatment of PIDs is given. Reducing the regrettable delay in diagnosis of PIDs (worldwide) is possible only when awareness is increased by doctors who may encounter patients with PID. This review shall serve this purpose. Progresses in understanding what the link might be between one genetic defect presenting in various phenotypes or how various gene defects may manifest by very similar PID phenotypes helps building awareness. Knowledge of PID favours early diagnosis, a cornerstone of optimal, sometimes life-long care at justifiable costs. The complexity of PIDs calls for clinical laboratory and clinical diagnostic performed by experts only. Exciting laboratory diagnostic progresses in early diagnosis of the most severe forms of PID are reviewed below. Progresses in curative therapies for PIDs, such as hematopoietic stem cell transplantation and gene therapies, are mentioned in short. About 80% of PID patients suffer from an antibody deficiency syndrome and can profit from non-curative replacement therapies with human immunoglobulin G concentrates. Modes of application, safety and hints for dosing of replacement therapies to reduce frequencies of severe infections are mentioned below. Thanks to the increasing quality of care, patients survive adolescence. A glance is given on the problems of transition to the adult medicine setting.

Sporadic late-onset nemaline myopathy with MGUS: long-term follow-up after melphalan and SCT.

OBJECTIVE Sporadic late-onset nemaline myopathy (SLONM) is a rare, late-onset myopathy that progresses subacutely. If associated with a monoclonal gammopathy of unknown significance (MGUS), the outcome is unfavorable: the majority of these patients die within 1 to 5 years of respiratory failure. This study aims to qualitatively assess the long-term treatment effect of high-dose melphalan (HDM) followed by autologous stem cell transplantation (SCT) in a series of 8 patients with SLONM-MGUS. METHODS We performed a retrospective case series study (n = 8) on the long-term (1-8 years) treatment effect of HDM followed by autologous SCT (HDM-SCT) on survival, muscle strength, and functional capacities. RESULTS Seven patients showed a lasting moderate-good clinical response, 2 of them after the second HDM-SCT. All of them had a complete, a very good partial, or a partial hematologic response. One patient showed no clinical or hematologic response and died. CONCLUSIONS This case series shows the positive effect of HDM-SCT in this rare disorder. Factors that may portend an unfavorable outcome are a long disease course before the hematologic treatment and a poor hematologic response. Age at onset, level and type of M protein (κ vs λ), and severity of muscle weakness were not associated with a specific outcome. CLASSIFICATION OF EVIDENCE This study provides Class IV evidence that for patients with SLONM-MGUS, HDM-SCT increases the probability of survival and functional improvement.

Comparative therapeutic value of post-remission approaches in patients with acute myeloid leukemia aged 40-60 years.

The preferred type of post-remission therapy (PRT) in patients with acute myeloid leukemia (AML) in first complete remission (CR1) is a subject of continued debate, especially in patients at higher risk of nonrelapse mortality (NRM), including patients >40 years of age. We report results of a time-dependent multivariable analysis of allogenic hematopoietic stem cell transplantation (alloHSCT) (n=337) versus chemotherapy (n=271) or autologous HSCT (autoHSCT) (n=152) in 760 patients aged 40-60 years with AML in CR1. Patients receiving alloHSCT showed improved overall survival (OS) as compared with chemotherapy (respectively, 57±3% vs 40±3% at 5 years, P<0.001). Comparable OS was observed following alloHSCT and autoHSCT in patients with intermediate-risk AML (60±4 vs 54±5%). However, alloHSCT was associated with less relapse (hazard ratio (HR) 0.51, P<0.001) and better relapse-free survival (RFS) (HR 0.74, P=0.029) as compared with autoHSCT in intermediate-risk AMLs. AlloHSCT was applied following myeloablative conditioning (n=157) or reduced intensity conditioning (n=180), resulting in less NRM, but comparable outcome with respect to OS, RFS and relapse. Collectively, these results show that alloHSCT is to be preferred over chemotherapy as PRT in patients with intermediate- and poor-risk AML aged 40-60 years, whereas autoHSCT remains a treatment option to be considered in patients with intermediate-risk AML.Leukemia advance online publication, 23 December 2014; doi:10.1038/leu.2014.332.

A mouse model of HIES reveals pro- and anti-inflammatory functions of STAT3.

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.

Outcome of aplastic anaemia in children. A study by the severe aplastic anaemia and paediatric disease working parties of the European group blood and bone marrow transplant.

This study analysed the outcome of 563 Aplastic Anaemia (AA) children aged 0-12 years reported to the Severe Aplastic Anaemia Working Party database of the European Society for Blood and Marrow Transplantation, according to treatment received. Overall survival (OS) after upfront human leucocyte antigen-matched family donor (MFD) haematopoietic stem cell transplantation (HSCT) or immunosuppressive treatment (IST) was 91% vs. 87% (P 0·18). Event-free survival (EFS) after upfront MFD HSCT or IST was 87% vs. 33% (P 0·001). Ninety-one of 167 patients (55%) failed front-line IST and underwent rescue HSCT. The OS of this rescue group was 83% compared with 91% for upfront MFD HSCT patients and 97% for those who did not fail IST up-front (P 0·017). Rejection was 2% for MFD HSCT and HSCT post-IST failure (P 0·73). Acute graft-versus-host disease (GVHD) grade II-IV was 8% in MFD graft vs. 25% for HSCT post-IST failure (P < 0·0001). Chronic GVHD was 6% in MFD HSCT vs. 20% in HSCT post-IST failure (P < 0·0001). MFD HSCT is an excellent therapy for children with AA. IST has a high failure rate, but remains a reasonable first-line choice if MFD HSCT is not available because high OS enables access to HSCT, which is a very good rescue option.

Successful transnasal delivery of stem cells in a rat model of perinatal hypoxic-ischemic brain injury

OBJECTIVE: New routes for cell transplantation into the brain need to be explored as intracerebral or intrathecal applications have a high risk to cause damage to the central nervous system. It has been hypothesized that transnasally administrated cells bypass the blood-brain barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and neural progenitor cells (NPC) to perinatal rats in a model of hypoxic-ischemic brain injury. STUDY DESIGN: Four-day-old Wistar rat pups, previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, either received WJ-MSC or green fluorescent protein-expressing NPC: The heads of the rat pups were immobilized and 3 ml drops containing the cells (50’000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600’000 cells. Animals were sacrificed 24h, 48h or 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin and sectioned. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSION: Our data show that transnasal delivery of WJ-MSC and NPC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials.

Combination therapy for multidrug-resistant cytomegalovirus disease

Multidrug-resistant (MDR) cytomegalovirus (CMV) emerged after transient responses to ganciclovir, foscarnet, and cidofovir in a CMV-seropositive recipient who underwent allogeneic hematopoietic stem cell transplantation from a CMV-seronegative donor. Experimental treatments using leflunomide and artesunate failed. Re-transplantation from a CMV-seropositive donor supported by adoptive transfer of pp65-specific T cells and maribavir was followed by lasting suppression. This case illustrates that successful MDR CMV therapy may require individualized multidisciplinary approaches. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The European Hematology Association Roadmap for European Hematology Research: a consensus document

Abstract The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at Euro 23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap. The EHA Roadmap identifies nine sections in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders. The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients. Received December 15, 2015. Accepted January 27, 2016. Copyright © 2016, Ferrata Storti Foundation

Bloodstream infection in paediatric cancer centres--leukaemia and relapsed malignancies are independent risk factors.

UNLABELLED In a prospective multicentre study of bloodstream infection (BSI) from November 01, 2007 to July 31, 2010, seven paediatric cancer centres (PCC) from Germany and one from Switzerland included 770 paediatric cancer patients (58% males; median age 8.3 years, interquartile range (IQR) 3.8-14.8 years) comprising 153,193 individual days of surveillance (in- and outpatient days during intensive treatment). Broviac catheters were used in 63% of all patients and Ports in 20%. One hundred forty-two patients (18%; 95% CI 16 to 21%) experienced at least one BSI (179 BSIs in total; bacteraemia 70%, bacterial sepsis 27%, candidaemia 2%). In 57%, the BSI occurred in inpatients, in 79% after conventional chemotherapy. Only 56 % of the patients showed neutropenia at BSI onset. Eventually, patients with acute lymphoblastic leukaemia (ALL) or acute myeloblastic leukaemia (AML), relapsed malignancy and patients with a Broviac faced an increased risk of BSI in the multivariate analysis. Relapsed malignancy (16%) was an independent risk factor for all BSI and for Gram-positive BSI. CONCLUSION This study confirms relapsed malignancy as an independent risk factor for BSIs in paediatric cancer patients. On a unit level, data on BSIs in this high-risk population derived from prospective surveillance are not only mandatory to decide on empiric antimicrobial treatment but also beneficial in planning and evaluating preventive bundles. WHAT IS KNOWN • Paediatric cancer patients face an increased risk of nosocomial bloodstream infections (BSIs). • In most cases, these BSIs are associated with the use of a long-term central venous catheter (Broviac, Port), severe and prolonged immunosuppression (e.g. neutropenia) and other chemotherapy-induced alterations of host defence mechanisms (e.g. mucositis). What is New: • This study is the first multicentre study confirming relapsed malignancy as an independent risk factor for BSIs in paediatric cancer patients. • It describes the epidemiology of nosocomial BSI in paediatric cancer patients mainly outside the stem cell transplantation setting during conventional intensive therapy and argues for prospective surveillance programmes to target and evaluate preventive bundle interventions.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration.

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.

Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression (r2 > 0.80). Over a similar range of HSV1-tk expression in vivo, the percent injected dose retained per gram of liver was 0–23% for ganciclovir and 0–3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.

Modifications of retinal neurons in a mouse model of retinitis pigmentosa

Animal models of retinitis pigmentosa include the rd mouse, in which a mutation of a rod-specific phosphodiesterase leads to the rapid loss of photoreceptors during the early postnatal life. Very little is known about changes occurring in inner retinal neurons after photoreceptor loss. These changes are important in view of the possibility of restoring vision in retinas with photoreceptor degeneration by means of cell transplantation or direct stimulation of inner layers. In this paper, we show that bipolar and horizontal cells of the rd mouse retina undergo dramatic morphological modifications accompanying photoreceptor loss, demonstrating a dependence of second order neurons on these cells. While describing modifications of the rd retina, we also provide quantitative information about neurons of the wild-type mouse retina, useful for future studies on genetically altered animals.