Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

Abstract The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

The role of WNT and mTOR in human memory T cell formation

Cancer is one of the world's leading causes of death with a rising trend in incidence. These epidemiologic observations underline the need for novel treatment strategies. In this regard, a promising approach takes advantage of the adaptive effector mechanisms of the immune system, using T lymphocytes to specifically target and destroy tumour cells. However, whereas current approaches mainly depend on short-lived, terminally differentiated effector T cells, increasing evidence suggests that long lasting and maximum efficient immune responses are mediated by low differentiated memory T cells. These memory T cells should display characteristics of stem cells, such as longevity, self-renewal capacity and the ability to continuously give rise to further differentiated effectors. These stem celllike memory T (TSCM) cells are thought to be of key therapeutic value as they might not only attack differentiated tumour cells, but also eradicate the root cause of cancer, the cancer stem cells themselves. Thus, efforts are made to characterize TSCM cells and to identify the signalling pathways which mediate their induction. Recently, a human TSCM cell subset was described and the activation of the Wnt-ß-catenin signalling pathway by the drug TWS119 during naive CD8+ T (TN) cell priming was suggested to mediate their induction. However, a precise deciphering of the signalling pathways leading to TSCM cell induction and an in-depth characterization of in vitro induced and in vivo occurring TSCM cells remain to be performed. Here, evidence is presented that the induction of human and mouse CD8+ and CD4+ TSCM cells may be triggered by inhibition of mechanistic/mammalian target of rapamycin (mTOR) complex 1 with simultaneously active mTOR complex 2. This molecular mechanism arrests a fraction of activated TN cells in a stem cell-like differentiation state independently of the Wnt-ß-catenin signalling pathway. Of note, TWS119 was found to also inhibit mTORCl, thereby mediating the induction of TSCM cells. Suggesting an immunostimulatory effect, the acquired data broaden the therapeutic range of mTORCl inhibitors like rapamycin, which are, at present, exclusively used due to their immunosuppressive function. Furthermore, by performing broad metabolic analyses, a well-orchestrated interplay between intracellular signalling pathways and the T cells' metabolic programmes could be identified as important regulator of the T cells' differentiation fate. Moreover, in vitro induced CD4+ TSCM cells possess superior functional capacities and share fate-determining key factors with their naturally occurring counterparts, assessed by a first-time full transcriptome analysis of in vivo occurring CD4+ TN cell, TSCM cells and central memory (TCM) cells and in vitro induced CD4+ TSCM cells. Of interest, a group of 56 genes, with a unique expression profile in TSCM cells could be identified. Thus, a pharmacological mechanism allowing to confer sternness to activated TN cells has been found which might be highly relevant for the design of novel T cell-based cancer immunotherapies.

Neurogenic subventricular zone stem/progenitor cells are notch1-dependent in their active but not quiescent state.

The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.

Thrombosis in paroxysmal nocturnal hemoglobinuria at a glance: a clinical review.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired stem cell disorder, with its primary clinical manifestations being hemolytic anemia, marrow failure and thrombophilia. Chronic hemolysis, failures of the fibrinolytic system, increased leukocyte-derived tissue factor levels in plasma, procoagulant microparticles generated through complement-mediated damage of platelets and venous endothelium are related to the acquired hypercoagulable state. Visceral thrombosis (including hepatic veins and mesenteric veins), cerebrovascular events and pulmonary embolism predict a poor outcome. Thrombosis is also associated with significant morbidity during pregnancy. Depending on the sites of thrombosis, a score-based probability to predict outcome can be assigned. Abdominal vein thromboses account for the majority of morbidity and mortality related to thrombosis, and time-dependent trends suggest that mortality rates tend to decline, with the advent of evolution of therapeutic and diagnostic strategies. In contrast, mortality rates from cerebrovascular events display no significant decline. Prompt diagnosis requires both clinical suspicion and sophisticated imaging techniques, along with multidisciplinary therapeutic intervention. In the eculizumab era, a significant reduction of thrombotic events was observed during therapy, and long-term follow up is needed to establish any benefit in rates and pattern of this complication. However, up to now, only bone marrow transplantation permanently abolishes the coagulation defect.

A proteomics approach to decipher the molecular nature of planarian stem cells.

Background In recent years, planaria have emerged as an important model system for research into stem cells and regeneration. Attention is focused on their unique stem cells, the neoblasts, which can differentiate into any cell type present in the adult organism. Sequencing of the Schmidtea mediterranea genome and some expressed sequence tag projects have generated extensive data on the genetic profile of these cells. However, little information is available on their protein dynamics. Results We developed a proteomic strategy to identify neoblast-specific proteins. Here we describe the method and discuss the results in comparison to the genomic high-throughput analyses carried out in planaria and to proteomic studies using other stem cell systems. We also show functional data for some of the candidate genes selected in our proteomic approach. Conclusions We have developed an accurate and reliable mass-spectra-based proteomics approach to complement previous genomic studies and to further achieve a more accurate understanding and description of the molecular and cellular processes related to the neoblasts.

IFN alpha activates dormant haematopoietic stem cells in vivo

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly reestablish homeostasis(1). The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFN alpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFN alpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFN alpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFN alpha/beta receptor (IFNAR)(2), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFN alpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFN alpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil(1,5), HSCs pre-treated (primed) with IFN alpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFN alpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFN alpha pathway in HSCs impairs their function, acute IFN alpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFN alpha on leukaemic cells(6,7), and raise the possibility for new applications of type I interferons to target cancer stem cells(8).

Myc's other life: stem cells and beyond.

Over the last three decades genetic and biochemical studies have revealed the pleiotropic effects of the Myc oncoprotein. While cell line studies have defined the intracellular processes regulated by Myc such as proliferation, differentiation, and metabolic growth, in vivo studies have confirmed these functions, and revealed roles in acquisition and maintenance of stem cell properties. These roles may be partially mediated by Myc's capacity to modify the chromatin landscape on a global scale. Myc also regulates numerous protein-coding transcripts, and many noncoding RNAs (rRNAs, tRNAs, and miRNAs). As Myc activity directly correlates with protein expression, further complexity is provided by post-translational modifications that regulate Myc in normal stem cells or deregulate it in malignant stem cells.

Thérapie cellulaire en cardiologie: bilan des premiers essais cliniques randomisés [Cell therapies in cardiology: results from the first randomized clinical trials]

Following acute myocardial infarction, necrotic cardiac tissue is replaced by scar leading to ventricular remodeling and pump failure. Transplantation of autologous bone marrow-derived cells into the heart, early post-infarct, aims to prevent ventricular remodeling. This strategy has been evaluated in four controlled, randomized clinical trials, which provided mixed results. A transient improvement in ventricular function was observed in one trial, and a modest improvement (the duration of which remains to be determined) in an additional trial, whereas two trials showed negative results. A modest benefit of bone marrow cell transplantation was also observed in patients with chronic ischemic heart disease. Despite mixed results reported so far, cell therapy of heart disease still is in its infancy and has considerable room for improvement.

Plasticity of fetal cartilaginous cells.

Tissue-specific stem cells found in adult tissues can participate in the repair process following injury. However, adult tissues, such as articular cartilage and intervertebral disc, have low regeneration capacity, whereas fetal tissues, such as articular cartilage, show high regeneration ability. The presence of fetal stem cells in fetal cartilaginous tissues and their involvement in the regeneration of fetal cartilage is unknown. The aim of the study was to assess the chondrogenic differentiation and the plasticity of fetal cartilaginous cells. We compared the TGF-β3-induced chondrogenic differentiation of human fetal cells isolated from spine and cartilage tissues to that of human bone marrow stromal cells (BMSC). Stem cell surface markers and adipogenic and osteogenic plasticity of the two fetal cell types were also assessed. TGF-β3 stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens, and variable levels of type X collagen. Although fetal cells showed the same pattern of surface stem cell markers as BMSCs, both type of fetal cells had lower adipogenic and osteogenic differentiation capacity than BMSCs. Fetal cells from femoral head showed higher adipogenic differentiation than fetal cells from spine. These results show that fetal cells are already differentiated cells and may be a good compromise between stem cells and adult tissue cells for a cell-based therapy.

An EORTC Phase II study of caspofungin as first-line therapy of invasive aspergillosis in haematological patients.

OBJECTIVES: Caspofungin was evaluated as first-line monotherapy of invasive aspergillosis (IA) in patients with haematological malignancies and undergoing autologous transplants. METHODS: Adults with proven or probable IA, defined strictly according to EORTC-MSG criteria, were eligible. Those with possible IA were enrolled, but were not evaluable for efficacy unless upgraded to proven/probable disease within 7 days of registration based on investigations performed within 48 h after enrolment. Caspofungin dosage was 70 mg (day 1) followed by 50 mg/day. The primary endpoint was the proportion of patients with complete or partial response at the end of caspofungin therapy in the modified intention to treat (MITT) group; secondary endpoints were response and survival at day 84 and safety. RESULTS: In the MITT group (n = 61), 75% of patients had cancer not in remission (relapsing or refractory), 85% were neutropenic at enrolment and 49% had a Karnofsky score of < or =50. At end of treatment, 1 and 19 patients had complete and partial response, respectively [success rate 33% (20/61)], 9 (15%) achieved stabilization and 31 (51%) had disease progression. One patient was not evaluable. The 6 and 12 week survival rates were 66% (40/61) and 53% (32/60), respectively. Baseline characteristics associated with survival at day 84 were an underlying disease in remission (not relapsing or refractory) and Karnofsky score. Recovery from neutropenia at the end of treatment was also significantly associated with survival. No serious drug-related adverse events or discontinuations due to drug-related adverse events were observed. CONCLUSIONS: Caspofungin provided an observed response rate compatible with the null hypothesis of a true response rate of < or =35%. Underlying disease-related factors had a major impact on results.

Interactions between cancer stem cells and their niche govern metastatic colonization.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Harvest site influences the growth properties of adipose derived stem cells.

The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.

Isolation of cardiovascular precursor cells from the human fetal heart.

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

Fibrinmatrix erhöht Adhärenz von regenerativen peripheren Nervenzellen [Fibrin matrix enhances adherence of peripheral nerve regenerative cells]

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.