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WO1997035605A1 - Manipulation de cellules mitotiquement actives de la region hippocampique du cerveau - Google Patents

Manipulation de cellules mitotiquement actives de la region hippocampique du cerveau Download PDF

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Publication number
WO1997035605A1
WO1997035605A1 PCT/CA1997/000197 CA9700197W WO9735605A1 WO 1997035605 A1 WO1997035605 A1 WO 1997035605A1 CA 9700197 W CA9700197 W CA 9700197W WO 9735605 A1 WO9735605 A1 WO 9735605A1
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WO
WIPO (PCT)
Prior art keywords
cells
growth factor
hippocampal region
hippocampal
brdu
Prior art date
Application number
PCT/CA1997/000197
Other languages
English (en)
Inventor
Samuel Weiss
Brent A. Reynolds
Original Assignee
Neurospheres Holdings Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neurospheres Holdings Ltd. filed Critical Neurospheres Holdings Ltd.
Priority to AU20199/97A priority Critical patent/AU2019997A/en
Publication of WO1997035605A1 publication Critical patent/WO1997035605A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]

Definitions

  • This invention relates to the in vitro and in vivo manipulation of the population of mitotically active cells which have been identified in the hippocampal region of the adult brain. More particularly, this invention is related to a method of directly manipulating the endogenous precursor cells of the hippocampal formation, in vivo, to induce them to divide, differentiate and migrate so as to augment hippocampal cell numbers in order to increase or alter synaptic efficacy or to replace dysfunctional hippocampal cells, or those lost to injury or disease.
  • This small percentage of undifferentiated neural cells exhibit the three main defining characteristics of stem cells: they are undifferentiated cells capable of proliferation, self-maintenance (i.e. capable of dividing without limit) and the production of a large number of differentiated, functional progeny. (See Potten & Loeffler, Development 110 (1990) pp. 1001-1020). Under suitable culture conditions, such as those disclosed in published PCT Application Nos. WO 93/01275, WO 94/10292, WO 94/16718 and WO 95/13364, the stem cells can be continuously proliferated and passaged resulting in large numbers of undifferentiated cells. Culture conditions can be changed to induce the differentiation of the stem cell progeny.
  • the neural stem cells are multipotent because a single stem cell can produce progeny that differentiated into the three types of differentiated cells of the CNS: astrocytes (types I and II), oligodendrocytes, and neurons.
  • astrocytes types I and II
  • oligodendrocytes oligodendrocytes
  • neurons oligodendrocytes
  • CVA cerebrovascular accident
  • mitoticaily active cells have been known for many years that a population of mitoticaily active cells in the mammalian dentate gyms retain the ability to generate neurons and glia well into post-natal life. However, as no mitoticaily active cells were reported outside the dentate gyrus, it was believed that mitoticaily active cells were absent from other regions of the adult hippocampus. As the hippocampal region is an integral component of memory and sensory integration and thus the ability to learn, it would be desirable to be able to induce precursor cells throughout the hippocampal region to generate new hippocampal cells to augment normal function or to repair a deficit caused by disease or injury.
  • the mitoticaily active cells of the hippocampal region also exhibit stem cell characteristics. Accordingly, the procedures set forth in WO 93/01275, WO 94/10292 and WO 95/13364, with respect to the in vitro and in vivo proliferation and use of multipotent neural stem cells and their progeny are applicable to the mitoticaily active cells of the hippocampal region identified in the present application.
  • the term "neural stem cell” refers to an undifferentiated neural cell that can be induced fo proliferate using the methods of Reynolds and Weiss disclosed in the related applications referenced above.
  • a neural stem cell is capable of self-maintenance, meaning that with each cell division, one daughter cell will also be a stem cell.
  • the non-stem cell progeny of a neural stem cell are termed "progenitor cells.”
  • the progenitor cells generated from a single multipotent neural stem cell are capable of differentiating into neurons, astrocytes (type I and type II) and oligodendrocytes.
  • the neural stem cell is “multipotent” because its progeny have multiple differentiative pathways.
  • neural progenitor cell refers to an undifferentiated cell derived from a neural stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type. For example, an O-2A cell is a glial progenitor cell that gives rise to oligodendrocytes and type II astrocytes, and thus could be termed a "bipotential" progenitor cell.
  • a distinguishing feature of a progenitor cell is that, unlike a stem cell, it has limited proliferative ability and thus does not exhibit self-maintenance.
  • precursor cells refers to the progeny of neural stem cells, and thus includes both progenitor cells and daughter neural stem cells.
  • the mitoticaily active cells of the hippocampal region can be induced to proliferate and differentiate in vitro using the same techniques described in the published PCT applications referenced above. Accordingly, the mitoticaily active cells of the hippocampal region are referred to herein as "hippocampal stem cells.”
  • the hippocampal stem cells and their progeny can be genetically modified using the procedures described in WO 94/16718. In Vivo Proliferation, Differentiation, and Genetic Modification of Hippocampal Stem Cells and Their Progeny
  • the hippocampal stem cells can be' induced to proliferate and their progeny induced to differentiate in vivo by administering to the host, any growth factor(s) or pharmaceutical composition that will induce proliferation and differentiation of the stem cells in vitro.
  • any growth factor(s) or pharmaceutical composition that will induce proliferation and differentiation of the stem cells in vitro.
  • suitable growth factors and pharmaceutical compositions are described in the published PCT applications referenced above.
  • the techniques described in the above- referenced published PCT applications for the proliferation, differentiation, and genetic modification of neural stem cells and their progeny in vitro can be adapted to in vivo techniques, to induce the proliferation, differentiation, and genetic modification of hippocampal stem cells.
  • hippocampal stem cells allows for augmentation of normal function as well as replacement of cells lost, due to injury or disease, thus obviating the need for transplanting foreign cells into a patient. Additionally, the cells can be modified or genetically engineered in vivo so that they express various biological agents useful in the treatment of neurological disorders.
  • Administration of growth factors can be done by any method, including injection cannula, transfection of cells with growth hormone-expressing vectors, injection, timed-release apparati which can administer substances at the desired site, and the like.
  • Pharmaceutical compositions can be administered by any method, including injection cannula, injection, oral administration, timed-release apparati and the like.
  • the hippocampal stem cells can be induced to proliferate and differentiate in vivo by induction with particular growth factors or pharmaceutical compositions which will induce their proliferation and differentiation. Therefore, this latter method circumvents the problems associated with transplantation and immune reactions to foreign cells.
  • Any growth factor can be used, particularly EGF, TGF ⁇ , FGF-1 , FGF-2, NGF, and combinations thereof.
  • Growth factors can be administered in any manner known in the art in which the factors may either pass through or by-pass the blood-brain barrier.
  • Methods for allowing factors to pass through the blood-brain barrier include minimizing the size of the factor, or providing hydrophobic factors which may pass through more easily.
  • Growth factors may be injected directly into the hippocampal region to induce hippocampal stem cell proliferation, or they may be administered to the lateral ventricle which is near the hippocampal region.
  • the hippocampal stem cell progeny can migrate into regions that have been damaged as a result of injury or disease.
  • Hippocampal stem cell progeny cultured in vitro can be used for the screening of potential neurologically therapeutic compositions using the techniques described in PCT published application no. WO 96/09543.
  • Immunocvtochemistrv Sections processed for BrdU were rinsed in washing solution (0.1 M PBS containing 0.02% sodium azide) for 20 minutes before any immunocylochemistry was performed. The sections were then incubated overnight at room temperature in a solution containing mouse monoclonal antisera to either Calbindin D-28K (Sigma, diluted 1 :1000), GFAP (Boehringer Mannheim, diluted 1:200), S-100 (Sigma, diluted 1:500), or NeuN (Gift from R. Mullen, diluted 1 :50) all diluted in 0.1 M PBS + 0.3% Triton + 10% normal goat serum.
  • the sections were then rinsed in washing solution (3 x 15 minutes) and incubated in anti-mouse IgG CY3 (Jackson, diluted 1:100) for 1 hour.
  • the sections were rinsed again in washing solution (3 x 15 minutes), then incubated in 1.0N HCL for 30 minutes at 60 °C to denature the DNA.
  • sections were rinsed in washing solution (3 x 15 minutes), and incubated overnight at room temperature in a primary rat monoclonal antibody (1:50) directed against single-stranded DNA containing BrdU (Seralab).
  • the sections were rinsed in washing solution (3 x 15 minutes), and incubated for 1 hour in anti-rat IgG FITC (Jackson diluted 1:100 in washing solution) at room temperature then rinsed and coverslipped under FluorSave (Calbiochem).
  • BrdU labeled cells were also present at day zero, not only in the dentate gyrus but also in all regions of Ammon's Horn and other areas of the hippocampal region, including the subiculum, entorhinal cortex and amygdala, amongst others (Fig. 1). Within the first three weeks the number of BrdU positive cells declined in all regions, most likely as a result of cell death and the diluting out of label by fast cycling cells such as progenitor cells. From 3 to 12 weeks, the "57"
  • BrdU positive cell count stabilized, suggesting the presence of either slower cycling cells or quiescent cells such as stem cells.
  • BrdU-labeled cells in the infra- and supra-pyramidal limbs and the subgranular zone of the dentate gyrus differentiated into cells with morphological and antigenic characteristics of neural cells.
  • Double-label immunocytochemistry demonstrated that BrdU-labeled cells differentiated into 1) cells with the morphological and antigenic characteristics of astrocytes (GFAP+BrdU; S-100+BrdU) and 2) cells with the morphological and antigenic characteristics of neurons (dual-label NeuN+BrdU; Calbindin+BrdU).
  • Figure 1 shows the decrease in the average number of BrdU-labeied cells in the dentate gyrus, CA1 and CA3 from day 0 to 12 weeks.
  • Table 1 shows the quantitative and phenotypic analysis of BrdU-labeled cells over time within the dentate gyrus and Ammon's Horn.
  • Figure 2 shows the average number of BrdU-labeled cells in the murine hippocampus over 24 weeks. After 3 weeks the number of BrdU-labeled cells stabilizes, indicating a slowly-cycling, relatively quiescent, stem cell population.
  • the Ad vector transduces the cells within the-regibn with high efficiency, regardless of proliferative state, and the vector itself functions episomally, thus avoiding potential problems inherent to random integration of exogenous DNA into the host genome, such as the induction of malignancy.
  • hippocampal region cells are protected from signals which would normally induce apoptosis such as oxidative stress, growth factor withdrawal and other physiological mechanisms.
  • EXAMPLE 4 Administration of substances to stem cells in the Hippocampal Region:

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé permettant la prolifération in vivo d'une cellule nerveuse souche pluripotente située dans la région hippocampique d'un mammifère. Ledit procédé consiste à administrer à la région hippocampique au moins un facteur de croissance qui induit la prolifération de la cellule souche, de sorte que cette dernière produit une descendance capable de se différencier en neurones et en cellules gliales. Ledit procédé peut être utilisé comme traitement des maladies, traumatismes ou troubles neurologiques qui affectent la région hippocampique du cerveau, tel un accident vasculaire cérébral.
PCT/CA1997/000197 1996-03-26 1997-03-26 Manipulation de cellules mitotiquement actives de la region hippocampique du cerveau WO1997035605A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20199/97A AU2019997A (en) 1996-03-26 1997-03-26 Manipulation of mitotically active cells of the hippocampal region of the brain

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1410096P 1996-03-26 1996-03-26
US60/014,100 1996-03-26

Publications (1)

Publication Number Publication Date
WO1997035605A1 true WO1997035605A1 (fr) 1997-10-02

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AU (1) AU2019997A (fr)
CA (1) CA2249417A1 (fr)
WO (1) WO1997035605A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022127A1 (fr) * 1996-11-15 1998-05-28 Neurospheres Holdings Ltd. Pretraitement a l'aide de facteurs de croissance afin de proteger le systeme nerveux central contre tout endommagement
US7514259B2 (en) 2000-02-11 2009-04-07 Schepens Eye Research Institute Isolation and transplantation of retinal stem cells
US10758572B2 (en) 2012-02-17 2020-09-01 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013364A1 (fr) * 1993-11-09 1995-05-18 Neurospheres Holdings Ltd. Modification et manipulation in situ de cellules souches du systeme nerveux central

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013364A1 (fr) * 1993-11-09 1995-05-18 Neurospheres Holdings Ltd. Modification et manipulation in situ de cellules souches du systeme nerveux central

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
C. VICARIO-ABEJÓN ET AL.: "FUNCTIONS OF BASIC FIBROBLAST GROWTH FACTOR AND NEUROTRPHINS IN THE DIFFERENTIATION OF HIPPOCAMPAL NEURONS.", NEURON, vol. 15, no. 1, 1 July 1995 (1995-07-01), CAMBRIDGE, MA, US, pages 105 - 114, XP002034680 *
F.H. GAGE ET AL.: "SURVIVAL AND DIFFERENTIATION OF ADULT NEURONAL PROGENITOR CELLS TRANSPLANTED TO THE ADULT BRAIN", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, December 1995 (1995-12-01), WASHINGTON US, pages 11879 - 11883, XP002034681 *
T.D. PALMER ET AL.: "THE ADULT RAT HIPPOCAMPUS CONTAINS PRIMORDIAL NEURAL STEM CELLS.", MOLECULAR AND CELLULAR NEUROSCIENCE, vol. 8, no. 6, 1997, SAN DIEGO, CA, US, pages 389 - 404, XP002034682 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022127A1 (fr) * 1996-11-15 1998-05-28 Neurospheres Holdings Ltd. Pretraitement a l'aide de facteurs de croissance afin de proteger le systeme nerveux central contre tout endommagement
US7514259B2 (en) 2000-02-11 2009-04-07 Schepens Eye Research Institute Isolation and transplantation of retinal stem cells
US10758572B2 (en) 2012-02-17 2020-09-01 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells
US11957719B2 (en) 2012-02-17 2024-04-16 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells

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Publication number Publication date
CA2249417A1 (fr) 1997-10-02
AU2019997A (en) 1997-10-17

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