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Enriched preparation of human fetal multipotential neural stem cells 実績あり

外国特許コード F110003093
整理番号 A011-02US
掲載日 2011年6月9日
出願国 アメリカ合衆国
出願番号 74781000
公報番号 20020012903
公報番号 7468277
出願日 平成12年12月22日(2000.12.22)
公報発行日 平成14年1月31日(2002.1.31)
公報発行日 平成20年12月23日(2008.12.23)
優先権データ
  • 60/173,003P (1999.12.23) US
発明の名称 (英語) Enriched preparation of human fetal multipotential neural stem cells 実績あり
発明の概要(英語) The present invention relates to a method of separating multipotential neural progenitor cells from a mixed population of cell types.
This method includes selecting a promoter which functions selectively in the neural progenitor cells, introducing a nucleic acid molecule encoding a fluorescent protein under control of said promoter into all cell types of the mixed population of cell types, allowing only the neural progenitor cells, but not other cell types, within the mixed population to express said fluorescent protein, identifying cells of the mixed population of cell types that are fluorescent, which are restricted to the neural progenitor cells, and separating the fluorescent cells from the mixed population of cell types, wherein the separated cells are restricted to the neural progenitor cells.
The present invention also relates to an isolated human musashi promoter and an enriched preparation of isolated multipotential neural progenitor cells.
従来技術、競合技術の概要(英語) BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis.
Full citations for these publications are provided at the end of the Detailed Description.
The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The damaged brain is largely incapable of functionally significant structural self-repair.
This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, 1980; Sturrock, 1982).
However, the absence of neuronal production in the adult vertebrate forebrain appears to reflect not a lack of appropriate neuronal precursors, but rather their tonic inhibition and/or lack of post-mitotic trophic and migratory support.
Converging lines of evidence now support the contention that neuronal and glial precursor cells are distributed widely throughout the ventricular subependymal of the adult vertebrate forebrain, persisting across a wide range of species groups (Goldman and Nottebohm, 1983; Reynolds and Weiss, 1992; Richards et al., 1992; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995a; reviewed in Goldman, 1995; Goldman, 1997; Goldman, 1998; Goldman and Luskin, 1998; and Gage et al., 1995).
Most studies have found that the principal source of these precursors is the ventricular zone (Goldman and Nottebohm, 1983; Goldman, 1990; Goldman et al., 1992; Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995), though competent neural precursors have been obtained from parenchymal sites as well (Richards et al., 1992; Palmer et al., 1995; Pincus et al., 1998).
In general, adult progenitors respond to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) with proliferative expansion (Reynolds and Weiss, 1992; Kilpatrick and Bartlett, 1995; Kuhn et al., 1997), may be multipotential (Vescovi et al., 1993; Goldman et al., 1996), and persist throughout life (Goldman et al., 1996).
In rodents and humans, their neuronal daughter cells can be supported by brain-derived neurotrophic factor (BDNF) (Kirschenbaum and Goldman, 1995a), and become fully functional in vitro (Kirschenbaum et al., 1994, Pincus et al., 1998a, and Pincus et al. 1998b), like their avian counterparts (Goldman and Nedergaard, 1992).
A major impediment to both the analysis of the biology of adult neural precursors, and to their use in engraftment and transplantation studies, has been their relative scarcity in adult brain tissue, and their consequent low yield when harvested by enzymatic dissociation and purification techniques.
As a result, attempts at either manipulating single adult-derived precursors or enriching them for therapeutic replacement have been difficult.
The few reported successes at harvesting these cells from dissociates of adult brain, whether using avian (Goldman et al., 1992; 1996c), murine (Reynolds and Weiss, 1992), or human (Kirschenbaum et al., 1994) tissue, have all reported <1% cell survival.
Thus, several groups have taken the approach of raising lines derived from single isolated precursors, continuously exposed to mitogens in serum-free suspension culture (Reynolds and Weiss, 1992; Morshead et al., 1994; Palmer et al., 1995).
As a result, however, many of the basic studies of differentiation and growth control in the neural precursor population have been based upon small numbers of founder cells, passaged greatly over prolonged periods of time, under constant mitogenic stimulation.
The phenotypic potential, transformation state and karyotype of these cells are all uncertain; after repetitive passage, it is unclear whether such precursor lines remain biologically representative of their parental precursors, or instead become transformants with perturbed growth and lineage control.
In order to devise a more efficient means of isolating native, unpassaged and untransformed progenitor cells from brain tissue, a strategy by which brain cells could be freely dissociated from brain tissue, then transduced in vitro with plasmid DNA bearing a fluorescent reporter gene under the control of neural progenitor cell-type specific promoters was developed (Wang et al., 1998).
This permitted isolation of the elusive neuronal progenitor cell of the CNS, using the Talpha 1 tubulin promoter, a regulatory sequence expressed only in neuronal progenitor cells and young neurons.
However, Talpha 1 tubulin-based separations are limited in that they yield committed neuronal progenitors, and not the more multipotential neural progenitors, such as neural stem cells, of the adult brain, which can give rise to neurons, oligodendrocytes, and astrocytes.
The existence of these neural stem cells has been reported in a number of studies of rodents (reviewed in Weiss et al., 1996), and precursors competent to generate both neurons and oligodendrocytes have been demonstrated in adult humans (Kirschenbaum et al., 1994; reviewed in Goldman, 1997).
In rodents, these cells have been clonally expanded using repetitive passage and mitogenic stimulation, as described above.
Nonetheless, native adult neural stem cells have never been separated and purified as such, in rodents or humans.
A strong need therefore exists for a new strategy for identifying, separating, isolating, and purifying native multipotential neural progenitor cells from brain tissue.

特許請求の範囲(英語) [claim1]
1. An enriched preparation of human fetal multipotential neural stem cells extracted directly from the brain of a human fetus without initial epidermal growth factor expansion, wherein the enriched preparation is capable of:
(1) generating neurons, astrocytes, and oligodendrocytes and (2) being propagated for at least 7 weeks, and wherein the enriched preparation comprises a transcriptionally active nestin enhancer or musashi promoter.
[claim2]
2. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the multipotential neural stem cells are from the ventricular zone.
[claim3]
3. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the multipotential neural stem cells are from the hippocampus.
[claim4]
4. The enriched preparation of human multipotential stem cells according to claim 1, wherein a musashi promoter, if present, is capable of functioning in all cells of the enriched preparation.
[claim5]
5. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the enriched or purified preparation of isolated human multipotential neural stem cells is capable of being propagated for at least 10 weeks.
[claim6]
6. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the preparation is a purified preparation of isolated human multipotential neural stem cells.
[claim7]
7. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the nestin enhancer, if present, is capable of functioning in all cells of the enriched or purified preparation.
[claim8]
8. The enriched preparation of human multipotential neural stem cells according to claim 1, wherein the multipotential neural stem cells are self-renewing.
  • 発明者/出願人(英語)
  • GOLDMAN STEVEN A
  • OKANO HIDEYUKI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • CORNELL RESEARCH FOUNDATION
国際特許分類(IPC)
米国特許分類/主・副
  • 435/368
  • 435/366
  • 435/377
  • 435/378
参考情報 (研究プロジェクト等) CREST Understanding the Brain AREA
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