Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2000-10-10
2003-12-23
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S366000, C435S377000, C435S320100, C536S023100
Reexamination Certificate
active
06667176
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the field of cell biology of embryonic cells. More specifically, it relates to the propagation of human pluripotent stem cells, culture conditions that facilitate propagation, and the use of such cultures for producing cDNA libraries.
BACKGROUND
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 as a result 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 pluripotent stem cells, which are thought to have the potential to differentiate into almost any cell type.
Early work on pluripotent stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). Mouse stem cells can be isolated both from early embryonic cells and germinal tissue. Desirable characteristics of pluripotent 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).
Expression libraries produced from mouse embryonic stem cells have been reported and the resulting sequences deposited in public databases (e.g., GenBank entries from the Washington University-Howard Hughes Medical Institute Mouse EST Project; RIKEN Institute Mouse ESTs). Typically, these libraries are constructed by first isolating mRNA from in vitro cultured mouse ES strains, then converting the mRNA to double-strand cDNA. Plasmid clones from such libraries are processed and DNA sequence is obtained from one or both ends of the cDNAs.
U.S. Pat. No. 5,789,158 (Knowles) reported production of cDNA libraries made from mRNA taken from unfertilized eggs, embryos, and 8-cell blastocysts. Subtraction libraries were reported from 2-cell and 8-cell stage embryos. Insert size of the cDNA in positive clones ranged from 500 to 900 base pairs. Adjaye et al. (Genomics 46:337, 1997) reported cDNA libraries formed from unfertilized oocytes, and embryos up to the blastocyst stage. The authors found that a high proportion of detected sequences were not found in the GenBank dbEST databases.
Nishiguichi et al. (J. Biochem. 119:749, 1996) used ESTs to identify genes expressed in mouse embryonal carcinoma F9 cells. Of 1026 randomly selected cDNA clones, 78% matched known genes, of which 53% were related to transcription and translation, and 19% were related to energy metabolism. Approximately 7% of the ESTs corresponding to low-abundance mRNAs were reported as related to retinoic acid-regulated genes, or mammalian development or differentiation related genes.
Sasaki et al (Genomics 49:167, 1998) reported gene expression in mouse blastocyst by single-pass EST sequencing of 3995 clones. Analysis of the cDNAs revealed that the library contained a variety of genes that did not match with homologs in the human EST database. The authors concluded that many early stage developmental genes remain to be identified.
Phillips et al. (Science 288:1635, 2000) analyzed the genetic program of hematopoietic stem cells. Subtracted cDNA libraries from purified murine fetal liver stem cells were analyzed with bioinformatic and array hybridization strategies. Several thousand previously undescribed gene products were determined, with properties suggestive of regulatory functions. Collected data on the molecular phenotype of the murine hematopoietic stem cell is available at the Princeton stem cell website.
Challenges in the Characterization and use of Human Pluripotent Stem Cells
The development of preparations of human pluripotent stem cells has involved overcoming a number of technical difficulties, and is considerably less advanced than work with mouse cells.
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent 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. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622).
Both hES and hEG cells have the long-sought characteristics of pluripotent stem cells: they are capable of long-term proliferation in vitro without differentiating, they have a normal karyotype, and they remain capable of producing a number of different cell types. Because of this, they hold considerable promise for use in human therapy, acting as a reservoir for regeneration of almost any tissue compromised by genetic abnormality, trauma, or a disease condition.
A significant challenge to the use of pluripotent stem cells for therapy is that they are traditionally cultured on a layer of feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780; U.S. Pat. No. 6,090,622). Without feeder fibroblasts in the culture environment, hPS cells soon die, or differentiate into a heterogeneous population of committed cells. Leukemia inhibitory factor (LIF) inhibits differentiation of mouse PS cells, but it does not replace the role of feeder cells in preventing differentiation of human PS cells. Unfortunately, using feeder cells increases production costs, impairs scale-up, and produces mixed cell populations that require the pluripotent stem cells be separated from feeder cell components.
Another challenge is to control differentiation of stem cells into the particular type of tissue required for treatment of each patient. It is a hypothesis of this invention that better understanding of the differentiation process will be obtained by observing gene expression during growth and differentiation of pluripotent stem cells.
International Patent Publication WO 99/20741 (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 which are either nucleosides or a pyruvate salt.
Sequence-based studies of early human development have focused on libraries produced from fetal organs and tissues for example, fetal libraries from the I.M.A.G.E. consortium. International Patent Publication WO 98/00540 (Incyte) reports partial sequences of stem cell antigens, isolated from cDNA libraries derived from THP-1 cells and bladder tumors.
As far as we know, successful production and characterization of expression libraries from human pluripotent stem cells or their derivatives has not previously been reported.
SUMMARY OF THE INVENTION
Described in this disclosure is a system for obtaining expression libraries from primate pluripotent stem (pPS) cells. pPS cells can be maintained in vitro without requiring a layer of feeder cells to inhibit differentiation. The role of the feeder cells is replaced by culture conditions that promote hPS cell growth without differentiation, exemplified by an extracellular matrix and conditioned medium. cDNA libraries from such cultures are substantially devoid of transcripts of feeder cell origin, relatively uncontaminated by transcripts from differentiated cells, and can have a high proportion of full-length transcripts. Subtraction libraries can also be produced that are enriched for transcripts modulated during differentiation.
So
Carpenter Melissa K.
Funk Walter D.
Gold Joseph D.
Inokuma Margaret S.
Xu Chunhui
Crouch Deborah
Earp David J.
Geron Corporation
Schiff J. Michael
Ton Thai-An N.
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