The 293 cell line was derived by transformation of primary cultures of human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA. A combination of immunostaining, immunoblot, and microarray analysis showed that 293 cells express the neurofilament (NF) subunits NF-L, NF-M, NF-H, and a-internexin as well as many other proteins typically found in neurons. Three other independently derived HEK lines, two transformed by Ad5 and one by Ad12, also expressed NFs, as did one human embryonic retinal cell line transformed with Ad5. Two rodent kidney lines transformed with Ad12 were also found to express NF proteins, although several rodent kidney cell lines transformed by Ad5 DNA and three HEK cell lines transformed by the SV40 early region did not express NFs. These results suggest that human Ads preferentially transform human neuronal lineage cells. We also demonstrate that the widely used HEK293 cells have an unexpected relationship to neurons, a finding that may require reinterpretation of many previous studies in which it was assumed that HEK293 cells resembled more typical kidney epithelial cells.

Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.

The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses Such particles possess the tropism of the virus from which the GP was derived For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virus-derived GPs Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity Particular attention is paid to publications of successfully targeting a specific organ or cell types

Altering the tropism of lentiviral vectors through pseudotyping.

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1–3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

http://callisto.ggsrv.com/imgsrv/FastFetch/UBER1/2IHT_nprot.2007.207-p

Isolation and culture of adult neurons and neurospheres

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

Gene Therapy Progress and Prospects: Development of improved lentiviral and retroviral vectors – design, biosafety, and production

Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering.

The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. T...

http://www.bristol.ac.uk/phys-pharm/media/virallab/coleman.pdf

Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo

Cloning and characterization of a secreted form of angiotensin-converting enzyme 2.

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to c...

https://www.physiology.org/doi/pdf/10.1152/jn.00088.2001

Characterization of Mitotic Neurons Derived From Adult Rat Hypothalamus and Brain Stem

Hypertension is a debilitating disease with significant socioeconomic and emotional impact. Despite recent success in the development of traditional pharmacotherapy for the management of hypertension, the incidence of this disease is on the rise and has reached epidemic proportions by all estimates. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau, and novel approaches for the treatment and control of hypertension must be explored. We have begun to investigate the possibility of treating and/or curing hypertension by using genetic means. In this review, we will provide evidence in favor of targeting of the renin-angiotensin system by antisense gene therapy as an effective strategy for the lifelong prevention of hypertension in the spontaneously hypertensive rat model. In addition, we will discuss the properties of an ideal vector for the systemic delivery of genes and the potential experimental hurdles that must be overcome to take this innovative approach to the next level of evaluation.

Matthew J. Huentelman

Papers

Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo

Cloning and characterization of a secreted form of angiotensin-converting enzyme 2.

Characterization of Mitotic Neurons Derived From Adult Rat Hypothalamus and Brain Stem

The Future of Hypertension Therapy: Sense, Antisense, or Nonsense?

HIV-1-based lentiviral vectors for use in vivo Efficient large-scale production and concentration of