Enumeration of Neural Stem Cells Using Clonal Assays.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages - astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions.

Advance chromatin extraction improves capture performance of protein A affinity chromatography.

Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise.

Chromatin-mediated depression of fractionation performance on electronegative multimodal chromatography media, its prevention, and ramifications for purification of immunoglobulin G.

Chromatin heteroaggregates comprising nucleosomal arrays, DNA, histone and non-histone host cell proteins contributed 28% of the host contaminant mass in an IgG cell culture harvest. A subset comprising soluble particles of 50-400nm was retained through its histone component by an electronegative multimodal chromatography adsorbent. Accumulated heteroaggregates hindered pore access and depressed IgG capacity by 69% compared with capacity determined with purified IgG.

Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance.

Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A.

Characterization and removal of aggregates formed by nonspecific interaction of IgM monoclonal antibodies with chromatin catabolites during cell culture production.

We observed that IgM monoclonal antibodies and aggregates in mammalian cell culture supernatants were associated nonspecifically with nucleosomes, DNA, and histone proteins derived from nuclei of host cells that died during antibody production. A series of multimodal sample treatments were evaluated for their ability to selectively remove these contaminants without significant antibody loss. The first consisted of adding 2,5-dioxo-4-imidazolidinyl urea (allantoin) and the DNA intercalating agent 7-ethoxyacridine-3,9-diamine (ethacridine), then flowing the supernatant through a column of mixed porous particles bearing metal affinity, anion exchange, and cation exchange functionalities.

Void exclusion of antibodies by grafted-ligand porous particle anion exchangers.

We describe a new variant of anion exchange chromatography in columns packed with porous particles that embody charged low-density polymer zones supported by a higher density polymer skeleton. IgG defies the norms of anion exchange and is excluded to the void volume at pH 3-10 and 0-4M NaCl. Void exclusion also occurs with Fab, F(ab')2, and IgM.

Immunoglobulins M survive low-pH conditions used for virus inactivation and for elution from bioaffinity columns.

Tolerance of low pH is crucial for recombinant proteins to survive conditions that might be experienced during manufacturing such as virus inactivation and elution from bioaffinity columns. In this study, we exposed three different purified immunoglobulins M (IgMs) to pH 3.5 for 60 min at room temperature.

Principles and applications of steric exclusion chromatography.

We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography.

DNA-dependent Oct4-Sox2 interaction and diffusion properties characteristic of the pluripotent cell state revealed by fluorescence spectroscopy.

Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements.

Identification of ApoE as an autocrine/paracrine factor that stimulates neural stem cell survival via MAPK/ERK signaling pathway.

Neural stem cells (NSCs) are self-renewing multipotent cells that undergo symmetric and asymmetric cell division during development of the nervous system. The behavior of NSCs is tightly regulated by intrinsic processes such as transcriptional and post-transcriptional control, as well as the stem cell niche factors that activate ligand-receptor-mediated signaling pathways. However, the role of these niche factors that regulate NSC behavior is not clearly understood.

CSPG is a secreted factor that stimulates neural stem cell survival possibly by enhanced EGFR signaling.

Understanding how autocrine/paracrine factors regulate neural stem cell (NSC) survival and growth is fundamental to the utilization of these cells for therapeutic applications and as cellular models for the brain. In vitro, NSCs can be propagated along with neural progenitors (NPs) as neurospheres (nsphs). The nsph conditioned medium (nsph-CM) contains cell-secreted factors that can regulate NSC behavior.

Transcription factors and neural stem cell self-renewal, growth and differentiation.

The central nervous system (CNS) is a large network of interconnecting and intercommunicating cells that form functional circuits. Disease and injury of the CNS are prominent features of the healthcare landscape. There is an urgent unmet need to generate therapeutic solutions for CNS disease/injury.