Comparison of lentiviral vector titration methods

BACKGROUND: Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression. RESULTS: We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes. CONCLUSION: The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others

Concise review: Therapeutic strategies for Parkinson disease based on the modulation of adult meurogenesis

Parkinson disease (PD) is a progressive neurodegenerative disorder affecting millions of people worldwide. To date, treatment strategies are mainly symptomatic and aimed at increasing dopamine levels in the degenerating nigrostriatal system. Hope rests upon the development of effective neurorestorative or neuroregenerative therapies based on gene and stem cell therapy or a combination of both. The results of experimental therapies based on transplanting exogenous dopamine-rich fetal cells or glial cell line-derived neurotrophic factor overexpression into the brain of Parkinson disease patients encourage future cell- and gene-based strategies. The endogenous neural stem cells of the adult brain provide an alternative and attractive cell source for neuroregeneration. Prior to designing endogenous stem cell therapies, the possible impact of PD on adult neuronal stem cell pools and their neurogenic potential must be investigated. We review the experimental data obtained in animal models or based on analysis of patients' brains prior to describing different treatment strategies. Strategies aimed at enhancing neuronal stem cell proliferation and/or differentiation in the striatum or the substantia nigra will have to be compared in animal models and selected prior to clinical studies.status: publishe

Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity

Comparison of the transcriptomes of pluripotent embryonic stem cells, multipotent adult progenitor cells and lineage restricted mesenchymal stem cells identified a unique gene expression profile of multipotent adult progenitor cells

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC):The Hot Start experience

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field

Upscaling of lentiviral vector production by tangential flow filtration

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.status: publishe

Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo

Modulation of adult neurogenesis may offer new therapeutic strategies for various brain disorders. In the adult mammalian brain the subventricular zone (SVZ) of the lateral ventricle is a region of continuous neurogenesis. Lentiviral vectors stably integrate into dividing and nondividing cells, in contrast to retroviral vectors, which integrate only into dividing cells. We compared their potential for gene transfer into both quiescent and slowly dividing stem cells as well as into more rapidly dividing progenitor cells. In contrast to retroviral vectors, stereotactic injection of lentiviral vectors into the SVZ of adult mice resulted in efficient and long-term marker gene expression in cells with characteristics of both immature type B cells and migrating precursor cells. After migration along the rostral migratory stream and differentiation, the number of enhanced green fluorescent protein (eGFP)-expressing granular and periglomerular interneurons increased over time in the ipsilateral olfactory bulb. Moreover, the number of eGFP-labeled neuronal progenitor cells in the SVZ increased over time. By intraventricular injection of lentiviral vectors we could restrict gene transfer to ependymal cells and type B astroglial-like stem cells. In conclusion, lentiviral vectors surpass retroviral vectors in efficient long-term in vivo marking of subventricular zone stem cells for basic research and therapeutic applications.status: publishe

Noninvasive and quantitative monitoring of adult neuronal stem cell migration in mouse brain using bioluminescence imaging

It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.status: publishe

Isolation procedure and characterization of multipotent adult progenitor cells from rat bone marrow

Multipotent adult progenitor cells (MAPCs) are adult stem cells derived from the bone marrow of mouse and rat and were described for the first time in 2002 (Jiang et al., Nature 418:41-49, 2002), and subsequently (Breyer et al., Exp Hematol 34:1596-1601, 2006; Jiang et al., Exp Hematol 30:896-904, 2002; Ulloa-Montoya et al., Genome Biol 8:R163, 2007). The capacity of rodent MAPC to differentiate at the single-cell level into some of the cell types of endoderm, mesoderm, and neuroectoderm germ layer lineages makes them promising candidates for the study of developmental processes. MAPC are isolated using adherent cell cultures and are selected based on morphology after a period of about 8-18 weeks. Here, we describe a step-by-step reproducible method to isolate rat MAPC from fetal and adult bone marrow. We elaborate on several aspects of the isolation protocol including, cell density and medium components, and methods for selecting and obtaining potential MAPC clones and their characterization.status: publishe