Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1998-11-18
2001-02-06
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S375000
Reexamination Certificate
active
06184035
ABSTRACT:
BACKGROUND OF THE INVENTION
Regeneration after injury in post-natal organ systems, in many ways, recapitulates developmental processes during embryogenesis. Though many interesting and crucial individual genes that are important for embryogenesis and organogenesis have been discovered over the past decade, the integrated regulation of the process is in many ways unknown (Barinaga, 1994), as are the similarities and differences between embryonic development and regeneration/healing of post-natal cells, tissues and organs. In embryonic mice and man, the various tissue systems develop in parallel and use both inter- and intra-tissue signaling, while the environment around the tissue progresses from one dependent on diffusion of oxygen to one in which oxygen is supplied via the developing vascular system. In the embryo over time, oxygenation to tissues increases as the blood supply is laid down and extended, but this delivery of oxygen is not homogenous throughout any tissue. And though oxygenation becomes richer as the embryo grows, levels of oxygen present in the embryo are generally considered insufficient for normal adult tissue functioning.
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. A similar course of coupled cell proliferation and differentiation in the presence of changing local O
2
supply occurs 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).
The hypothesis that O
2
levels have significant differential impact on different cell types or states has so far received little explicit attention in the literature, with the exception of formation of the vasculature itself. In particular it is important to note that the vast preponderance of studies of regeneration in vitro are performed in laboratories using room air oxygen levels. In room air, 20-21% of atmospheric gas is oxygen (at sea level depending on humidity), which translates into an oxygen partial pressure of 160 mm Hg [0.21(760 mm Hg)]. The most highly oxygenated tissue in the human body is the arterial blood supply with an oxygen partial pressure of 90 mm Hg. Normal venous oxygenation is 40 mm Hg, and mean tissue oxygen level is 26 mm Hg. However, the vast majority of regeneration research or research on the culture of progenitor cells, stem cells, or differentiating products ignores the importance of oxygenation: The average tissue culture condition is 21% oxygen and 5% carbon dioxide which the remainder being nitrogen.
Herein, the inventors demonstrate that regulated oxygen levels, particularly subatmospheric levels of oxygen (i.e. levels below 21% oxygen and 5% carbon dioxide), can be used to exploit responses of stem and progenitor cells that differ from the response of other cells as a simple and general pathway for their isolation, maintenance, proliferation, enrichment, and/or selective developmental progression and differentiation. This work has important implications for clinical tissue and organ transplantation.
In a time of critical shortages of donor organs, efforts to bring cellular transplantation into the clinical arena are urgently needed (Neelakanta & Csete, 1996). For example, in the case of the liver, a stem cell has not been rigorously identified, and animal models of transplantation of fully-differentiated liver cells (normally quiescent and difficult to force into division experimentally) are not yet successful enough to warrant clinical trials. However, a liver stem cell would represent the ideal cellular transplant because of the potential to regenerate substantial organ function from a tiny rudiment. Thus, there remains a need for methods to identify cells (progenitors and stems) which can be used to regenerate tissue. The present invention is directed at these goals.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of isolating, maintaining, and/or enriching for stem or progenitor cells derived from diverse organ or tissue sources. The method can also be used to influence specific differentiation pathways, to alter the kinetics of developmental progression or differentiation or to alter the yield of one cell phenotype over other possible types via the use of subatmospheric or sub-physiologic oxygen levels. The invention specifically teaches that these can be accomplished by the controlled use of subatmospheric oxygen culture, and that the precise oxygen level or levels must be determined empirically and/or by reference to physiologic levels within intact functioning organ or tissue, or that tissue following injury, regeneration, or disease.
The inventors have discovered that when adult skeletal muscle fibers are cultured under physiologically hypoxic conditions, progenitor cells (which represent a source of regenerated new muscle) develop in greater numbers than when fibers are grown under traditional room air conditions. Furthermore, this subatmospheric oxygen-induced enrichment of the skeletal muscle progenitor population is followed by earlier regeneration of new skeletal muscle in culture.
Intentional hypoxia has been used in some culture systems, usually differentiated systems, to mimic pathologic conditions such as stroke (for example, Papadopoulos et al., 1996). In some circumstances, whole embryos have been cultured under subatmospheric oxygen conditions to mimic gestation (for example, Giles & Foote, 1997). In other circumstances reduced oxygen conditions are used as part of a broad range of tests of many environmental parameters on culture integrity (for example, Berthelot & Terqui, 1996). In a few cases oxygen conditions have been used to assess proliferation of particular cells as a function of oxygen levels (for example, Matsuda et al., 1998). However, such examples do not teach the general principle that an intentional reduction of oxygen surrounding cultures can be used to selectively promote survival, proliferation, enrichment or particular developmental or differentiation pathways from stem cell and/or progenitor cells. Furthermore, such reports have not had an effect on the general state or practice of the art, in that almost all tissue culture continues to be conducted in room air.
DETAILED DESCRIPTION OF THE INVENTION
A method for isolating, maintaining, propagating or enriching progenitor or stem cells, and/or for influencing the differentiation outcome or differentiation kinetics of such cells into particular tissue types, comprising the steps of:
a) obtaining cells derived from mammalian tissue containing at least one progenitor cell or stem cell capable of producing progeny that can assume or produce cells with one or more differentiated phenotypes, and
b) culturing the cells derived from such mammalian tissue (various organ and tissue types) in suitable medium under empirically determined subatmospheric oxygen conditions for a time sufficient to promote the survival, proliferation, or enrichment of the stem or progenitor population, or to cause or influence entry into one or more differentiation pathways.
Definitions
A “stem cell” is a relatively undifferentiated cell that can be induced to proliferate and that can produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In many biological instances, st
Csete Marie
Doyle John
Wold Barbara
Afremova Vera
Brinks Hofer Gilson & Lione
California Institute of Technology
Saucier Sandra E.
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