Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2001-07-06
2003-11-04
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S374000, C435S363000, C435S383000, C435S384000
Reexamination Certificate
active
06642048
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the field of cell biology of embryonic cells. More specifically, it relates to the derivation of cell lines producing conditioned culture media, which in turn can be used to propagate human pluripotent stem cells in feeder-free culture.
BACKGROUND
A number of recent discoveries have raised expectations that stem cells may be a source of replacement cells and tissue for cells and tissues that are damaged in the course of disease, infection, or because of congenital abnormalities. Various types of putative stem cells differentiate when they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, the liver, or the brain.
A particularly important discovery has been the development of human pluripotent stem (hPS) cells (reviewed by R. A. Pedersen, Scientif. Am. 280(4):68, 1999). These cells have the capacity to differentiate into essentially all types of cells in the body. For example, hPS cells have been used to generate cells that are committed to a number of different cell lineages, which retain their capacity to proliferate. Since these embryonic cells are truly pluripotent, they have the potential to provide a stock supply of different types of cells for regeneration of essentially any type of failed tissue.
Early work on embryonic stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). Desirable characteristics of stem cells are that they be capable of indefinite proliferation in vitro in an undifferentiated state, retain a normal karyotype, and retain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm).
The usual method for culturing mouse stem cells is to grow them on a layer of embryonic fibroblast feeder cells, such as the STO cell line. Cells from a previous culture are dispersed into a single cell suspension and cultured on the feeder cell layer. In some experimental procedures, it is possible to grow the mouse stem cells on gelatin plates without feeder cells, using recombinant soluble leukemia inhibitory factor (LIF) to replace the activity normally provided by the STO feeder cells (Robertson, supra). The presence of LIF is sufficient to keep the mouse stem cells from differentiating. By continuous passage of mouse stem cells, Berger et al. (Growth Factors 14:145,1997) selected out several cell lines independent of exogenously added LIF.
The morphology, cell surface markers, and growth requirements of embryonic cells from other species differ significantly from mouse embryonic stem cells. Stem cells from primate embryos are fragile, and it has been difficult to determine conditions that allow them to grow in culture. Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995) were the first to successfully culture stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. U.S.A. 95:13726, 1998 and International Patent Publication WO 98/43679). Both hES and hEG cells have the long-sought characteristics of hPS cells: they are capable of long-term proliferation in vitro without differentiating, they retain a normal karyotype, and they retain the capacity to differentiate to a number of different derivatives.
A major obstacle to the use of such cells in human therapy is that the originally described methods to propagate them involved culturing on a layer of feeder cells. For example, when cultured in a standard culture environment in the absence of mouse embryonic fibroblasts as feeder cells, the hPS cells rapidly differentiate or fail to survive. Unlike mouse ES cells, the presence of exogenously added LIF does not prevent differentiation of the human ES cells. Unfortunately, the use of feeder cells substantially increases the cost of production, and makes scale-up of hPS cell culture impractical. The feeder cells are metabolically inactivated to keep them from outgrowing the stem cells, so it is necessary to have fresh feeder cells for each splitting of the hES culture. Furthermore, procedures are not yet developed for completely separating feeder cell components away from embryonic cells prepared in bulk culture. When embryonic cells are grown using currently available culture methods, the presence of xenogeneic components from the feeder cells complicates their potential use in human therapy.
International Patent Publication WO/9920741 (Geron Corp.) is entitled “Methods and Materials for the Growth of Primate-Derived Primordial Stem Cells”. In one embodiment, a cell culture medium is provided for growing primate-derived primordial stem cells in a substantially undifferentiated state, having a low osmotic pressure and low endotoxin levels. The basic medium is combined with a nutrient serum effective to support the growth of primate-derived primordial stem cells and a substrate of feeder cells or an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and growth factors that are either nucleosides or a pyruvate salt.
New technology to facilitate growing undifferentiated hPS cells without feeders would be a major achievement towards realizing the full potential of embryonic cell therapy. In particular, there is a need for media that support feeder-free culture that is compatible with regulatory requirements for human administration, and can be scaled up for commercial production.
SUMMARY
This invention provides media that support the growth of primate pluripotent stem (pPS) cells in feeder-free culture, and cell lines useful for production of such media.
One embodiment of this invention is a method for preparing a conditioned medium suitable for culturing pPS cells in a growth environment essentially free of feeder cells, comprising conditioning medium by culturing cells in the medium, and then harvesting the conditioned medium.
Another embodiment of this invention is a method for culturing pPS cells, comprising providing a conditioned medium of this invention, and culturing pPS cells in a feeder-free growth environment containing the conditioned medium. In either of these embodiments, the pPS cells can be human embryonic stem (hES) cells.
The cells used for conditioning the medium are taken from a cell line that can proliferate in culture for an extended period, typically at least about 60 days. This does not necessarily mean that the particular cells used for conditioning the medium be long-lived; they can be irradiated or otherwise adapted to limit replication. However, the parent cell line from which they are taken will have the proliferative capacity stated. In particular embodiments, the cell line is an immortalized mouse cell line, or a human cell line, and may have characteristics of a fibroblast or muscle cell line. Exemplary lines are obtained by differentiating human embryonic stem cells ex vivo, and are euploid. Any of these cell lines can be genetically altered to express a growth factor (such as bFGF) or telomerase reverse transcriptase (TERT) at an elevated level.
Another embodiment of this invention is conditioned medium to support culturing pPS cells in a growth environment essentially free of feeder cells, prepared according to a method of this invention. A further embodiment is a composition of proliferating pPS cells or other cells in a growth environment that includes the conditioned medium of this invention, optionally free of feeder cells.
Yet another embodiment of this invention is a human cell line obtained by differentiating a culture of human embryonic stem (hES) cells into a population of differentiated cells that comprises fibroblast-like or mesenchymal cells, and then selecting such cells from the culture. Medium conditioned by culturing with the selected cells supports growth of pPS c
Gold Joseph D.
Xu Chunhui
Crouch Deborah
Earp David J.
Geron Corporation
Schiff J Michael
Ton Thai-An N.
LandOfFree
Conditioned media for propagating human pluripotent stem cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Conditioned media for propagating human pluripotent stem cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Conditioned media for propagating human pluripotent stem cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3177732