Lineage-restricted neuronal precursors and methods of isolation

Details Lineage-restricted neuronal precursors and methods of isolation Lineage-restricted neuronal precursors and methods of isolation

1998-07-02

2004-09-07

Kunz, Gary (Department: 1647)

Chemistry: molecular biology and microbiology

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

Primate cell, per se

C435S377000

Reexamination Certificate

active

06787353

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to lineage-restricted intermediate precursor cells and methods of making and using thereof. More particularly, the invention relates to neuronal-restricted precursors (NRP's) isolated from mammalian embryos, neuroepithelial stem (NEP) cells, or embryonic stem (ES) cells. These neuronal-restricted precursors are capable of self-renewal and differentiation into neurons, but not into glia, i.e. astrocytes and oligodendrocytes. Methods of generating, isolating, culturing, transfecting, and transplanting such neuronal-restricted precursor cells are also described.
Multipotent cells with the characteristics of stem cells have been identified in several regions of the central nervous system and at several developmental stages. F. H. Gage et al., Isolation, Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev. Neurosci. 159-92 (1995); M. Marvin & R. McKay, Multipotential Stem Cells in the Vertebrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, The Lineages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). These cells, often referred to as neuroepithelial stem cells (NEP cells), have the capacity to undergo self renewal and to differentiate into neurons, oligodendrocytes, and astrocytes, thus representing multipotent stem cells. A. A. Davis & S. Temple, A Self-Renewing Multipotential Stem Cell in Embryonic Rat Cerebral Cortex, 362 Nature 363-72 (1994); A. G. Gritti et al., Multipotential Stem Cells from the Adult Mouse Brain Proliferate and Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J. Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A Multipotent EGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci. 4565-74 (1992); B. A. Reynolds & S. Weiss, Clonal and Population Analyses Demonstrate that an EGF-Responsive Mammalian Embryonic CNS Precursor is a Stem Cell, 175 Developmental Biol. 1-13 (1996); B. P. Williams et al., The Generation of Neurons and Oligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 (1991).
The nervous system also contains precursor cells with restricted differentiation potentials. T. J. Kilpatrick & P. F. Bartlett, Cloned Multipotential Precursors from the Mouse Cerebrum Require FGF-2, Whereas Glial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); J. Price et al., Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer, 84 Developmental Biol. 156-60 (1987); B. A. Reynolds et al., supra; B. A. Reynolds & S. Weiss, supra; B. Williams, Precursor Cell Types in the Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P. Williams et al., supra. The relationship between multipotent stem cells and lineage restricted precursor cells is still unclear. In principal, lineage restricted cells could be derived from multipotent cells, but this is still a hypothetical possibility in the nervous system with no direct experimental evidence. Further, no method of purifying such precursors from multipotent cells has been described.
As has been shown in copending U.S. patent application Ser. No. 08/852,744, entitled “Generation, Characterization, and Isolation of Neuroepithelial Stem Cells and Lineage Restricted Intermediate Precursor,” filed May 7, 1997, hereby incorporated by reference in its entirety, NEP cells grow on fibronectin and require fibroblast growth factor (FGF) and an as yet uncharacterized component present in chick embryo extract (CEE) to proliferate and maintain an undifferentiated phenotype in culture. The growth requirements of NEP cells are different from neurospheres isolated from E14.5 cortical ventricular zone cells. B. A. Reynolds et al., supra; B. A. Reynolds & S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; WO 9409119. Neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on epidermal growth factor (EGF) for survival. FGF itself is not sufficient for long term growth of neurospheres, though FGF may support their growth transiently. NEP cells, however, grow in adherent culture, are FGF dependent, do not express detectable levels of EGF receptors, and are isolated at a stage of embryonic development prior to which it has been possible to isolate neurospheres. Thus, NEP cells may represent a multipotent precursor characteristic of the brain stem and spinal cord, while neurospheres may represent a stem cell more characteristic of the cortex. Nonetheless, NEP cells provide a model system for studying the principles of lineage restriction from multipotent stem cells or precursor cells of the central nervous system. The principles elucidated from the study of NEP cells are expected to be broadly applicable to all CNS precursor cells sufficiently multipotent to generate both neurons and glia. Thus, the present application is intended to be applicable to any CNS precursor cells regardless of their site of derivation as long as they are able to differentiate to both neurons and glial cells.
U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L. Stemple, discloses mammalian neural crest stem cells and methods of isolation and clonal propagation thereof, but fails to disclose cultured NEP cells, cultured lineage restricted precursor cells, and methods of generating, isolating, and culturing thereof. Neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas the neuroepithelial stem cells differentiate into neurons and glia of the central nervous system (CNS).
U.S. Ser. No. 08/909,435, filed Jul. 4, 1997, for “Isolation of Lineage Restricted Neuronal Precursors,” hereby incorporated by reference in its entirety, describes neuronal restricted precursor (NRP) cells that are capable of differentiating into neurons, but not into glial cells. It was shown that NRP cells can be isolated from NEP cells, as well as directly from embryonic spinal cords.
U.S. Ser. No. 08/980,850, filed Nov. 29, 1997, for “Lineage Restricted Glial Precursors from the Central Nervous System,” hereby incorporated by reference in its entirety, describes glial restricted precursor (GRP) cells that are capable of differentiating into oligodendrocytes, A2B5
+
process-bearing astrocytes, and A2B5
−
fibroblast-like astrocytes, but not into neurons. GRP cells can be isolated from differentiating NEP cells, as well as CNS tissue, and differ from oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells in growth factor requirements, morphology, and progeny.
In U.S. patent application Ser. No. 09/073,881, filed May 6, 1998, for “Common Neural Progenitor for CNS and PNS,” hereby incorporated by reference in its entirety, it was shown that NEP cells can be induced to differentiate into neural crest cells as well as other cells of the CNS and PNS.
The neuron-restricted precursor cells described herein are distinct from the NEP cells, GRP cells, neurospheres, and neural crest stem cells that have been described elsewhere. NEP cells are capable of differentiating into neurons or glia whereas NRPs can differentiate into neurons, but not glia, and NEP cells and NRPs display distinct cell markers. GRP cells can differentiate into glia, but not neurons. As mentioned above, neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on EGF for survival, whereas NRP cells grow in adherent culture and do not express detectable levels of EGF receptors. Further, neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas NRP cells differentiate into neurons of the central nervous system (CNS). NRP cells express polysialated or embryonic neural cell adhesion molecule (E-NCAM), but NEP cells, neurospheres, GRP cells, and neural crest cells do not. Therefore, NRP cells are different in their proliferative potential, expression of cell markers, and nutritional requirements from these other cell types.
The ability to isolate and grow mammalian neuronal-restricted precursor cells in vitro

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