Low oxygen culturing of central nervous system progenitor cells

Details Low oxygen culturing of central nervous system progenitor cells Low oxygen culturing of central nervous system progenitor cells

1999-10-22

2003-08-26

Kemmerer, Elizabeth (Department: 1647)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S004000, C435S325000, C435S377000, C435S352000, C435S368000

Reexamination Certificate

active

06610540

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the growth of cells in culture. More particularly, the present invention provides methods and compositions for increasing cell survival, cell proliferation and/or cell differentiation along specific pathways by growing the cells in low ambient oxygen conditions.
BACKGROUND OF THE INVENTION
In a time of critical shortages of donor organs, efforts to bring cellular transplantation into the clinical arena are urgently needed (Neelakanta & Csete, 1996). Indeed, cellular and tissue transplantation is now well recognized as a desirable technique for the therapeutic intervention of a variety of disorders including cystic fibrosis (lungs), kidney failure, degenerative heart diseases and neurodegenerative disease. However, although this may be a desirable and much needed intervention, a major impediment to this type of therapeutic intervention is the lack of an available supply of viable, differentiated cells. Generally differentiated cells cannot be readily expanded in culture. Thus, methods of increasing the number and/or availability of differentiated, viable cells are needed.
The central nervous system (CNS) (brain and spinal cord) has poor regenerative capacity which is exemplified in a number of neurodegenerative disorders, such as Parkinson's Disease. Although such diseases can be somewhat controlled using pharmacological intervention (L-dopa in the case of Parkinson's Disease), the neuropathological damage and the debilitating progression is not reversed. Cell transplantation offers a potential alternative for reversing neuropathological damage as opposed to merely treating the consequences of such damage.
Cultured CNS stem cells can self-renew, and after mitogen withdrawal, have an intrinsic capacity to generate oligodendrocytes, astrocytes, and neurons in predictable proportions (Johe et al., 1996). Manipulation of this intrinsic differentiation capacity in culture has been used to define a complex array of factors that maintain, amplify, or diminish a particular differentiated phenotype. Most such studies emphasize a primary role for transcription factors in defining CNS lineage identity, as well as growth and trophic factors acting locally and over long distances (Johe et al., 1996, Panchinsion et al., 1998). Dopaminergic neurons and their progenitors from these cultures are of special interest as potential sources of replacement cellular therapies for Parkinson's Disease patients (reviewed in Olanow et al., 1996).
Ideally, ex vivo culture conditions should reproduce the in vivo cellular environment with perfect fidelity. This ideal is especially pertinent when explants are used to study development, because conditions may be defined for cell fate choice and differentiation. For CNS stem cell cultures, in particular, maximizing survival, proliferation, and cell fate choice leading to dopaminergic neurons is essential for future cellular transplant therapies. Thus, understanding and control of the differentiation of such cells is crucial for providing a viable, useful product that can be used in transplantation or for studying the behavior of CNS cells, in vitro, in response to various conditions.
In embryogenesis, each tissue and organ develops by an exquisitely organized progression in which relatively unspecialized or “undifferentiated” progenitor or stem cells give rise to progeny that ultimately assume distinctive, differentiated identities and functions. Mature tissues and organs are composed of many types of differentiated cells, with each cell type expressing a particular subset of genes that in turn specifies that cell's distinctive structure, specialized function, and capacity to interact with and respond to environmental signals and nutrients. These molecular, structural and functional capacities and properties comprise the cell phenotype. Similarly, coupled cell proliferation and/or differentiation occurs, in the presence of changing local O
2
supply, when an injured or degenerating adult tissue undergoes repair and regeneration. The level of oxygen is especially pertinent in many regeneration paradigms in which normal blood supply is reduced or even transiently stopped by trauma or embolic events (myocardial infarction, stroke and the like).
Therefore, in clinical settings, gases are appreciated as a primary variable in organ survival, with oxygen as the critical gas parameter. Virtually all modern cell culture is conducted at 37° C. in a gas atmosphere of 5% CO
2
and 95% air. These conditions match core human body temperature and approximate quite well physiologic CO
2
concentrations. For example, mean brain tissue CO
2
is 60 mm Hg or about 7% (Hoffman et al., 1998). However, in striking contrast, oxygen in standard tissue culture does not reflect physiologic tissue levels and is, in fact, distinctly hyperoxic.
At sea level, (unhumidified) room air contains 21% O
2
which translates into an oxygen partial pressure of 160 mm Hg [0.21(760 mm Hg)]. However, the body mean tissue oxygen levels are much lower than this level. Alveolar air contains 14% oxygen, arterial oxygen concentration is 12%, venous oxygen levels are 5.3%, and mean tissue intracellular oxygen concentration is only 3% (Guyton, and Hall, 1996). Furthermore, direct microelectrode measurements of tissue O
2
reveal that parts of the brain normally experience O
2
levels considerably lower than total body mean tissue oxygen levels, reflecting the high oxygen utilization in brain. These studies also highlight considerable regional variation in average brain oxygen levels (Table 1) that have been attributed to local differences in capillary density. Mean brain tissue oxygen concentration in adult rates is 1.5% (Silver and Erecinska, 1988), and mean fetal sheep brain oxygen tension has also been estimated at 1.6% (Koos and Power, 1987).
TABLE 1
Regional rat brain tissue partial pressures of oxygen measured by
microelectrode
Brain area
% O
2
Cortex (gray)
2.5-5.3
Cortex (white)
0.8-2.1
Hypothalamus
1.4-2.1
Hippocampus
2.6-3.9
Pons, fornix
0.1-0.4
Adapted from Silver, L, Erecinska, M. Oxygen and ion concentrations in normoxic and hypoxic brain cells. In
Oxygen Transport to Tissue XX,
7-15, edited by Hudetz and Bruley, Plenum Press, New York (1988).
Thus, from the discussion above it is clear that under standard culture conditions, the ambient oxygen levels are distinctly hyperoxic, and not at all within physiologic ranges. These conditions of cell growth are have been historically inadequate for generating cells and tissues for transplantation into the brain or other area of the body or for providing an accurate in vitro model of what is occurring in vivo. Thus, there remains a need for methods to produce differentiated cells which can be used for therapeutic and research purposes. The present invention is directed to providing such methods.
BRIEF SUMMARY OF THE INVENTION
The present invention is directed to growing cells in low ambient oxygen conditions in order to mimic the physiological oxygen conditions with greater fidelity. The growth of these cells in such conditions provides certain surprising and unexpected results. These results are exploited and described in further detail herein. More particularly, the present invention describes methods that may independently be useful in increasing cell survival, cell proliferation and/or cell differentiation along specific pathways.
In specific embodiments, the present invention describes a method of increasing cell differentiation comprising culturing undifferentiated central nervous system (CNS) cells in low ambient oxygen conditions, wherein the low ambient oxygen conditions promotes the cellular differentiation of the neuronal cells. The definitions of low ambient oxygen conditions are described in depth elsewhere in the specification. However, it is contemplated that in specific embodiments the low ambient oxygen conditions comprise an ambient oxygen condition of between about 0.25% to about 18% oxygen. In other embodiments, the ambient oxygen conditions compris

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