WO2002096439A1 - Use of haematopoietic stem cells in the generation of neural stem cells - Google Patents

Use of haematopoietic stem cells in the generation of neural stem cells Download PDF

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WO2002096439A1
WO2002096439A1 PCT/ES2002/000253 ES0200253W WO02096439A1 WO 2002096439 A1 WO2002096439 A1 WO 2002096439A1 ES 0200253 W ES0200253 W ES 0200253W WO 02096439 A1 WO02096439 A1 WO 02096439A1
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stem cells
hematopoietic stem
cells
neural
neural stem
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PCT/ES2002/000253
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Spanish (es)
French (fr)
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Pedro ALARCÓN MARTÍNEZ
Sonia BONILLA JIMÉNEZ
Augusto Gerardo SILVA GONZÁLEZ
Salvador MARTÍNEZ PÉREZ
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Universidad Miguel Hernandez De Elche
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Priority to IL15435602A priority Critical patent/IL154356A0/en
Publication of WO2002096439A1 publication Critical patent/WO2002096439A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

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  • the invention relates to the generation of neural stem cells, both in vitro and in vivo, from hematopoietic stem cells.
  • Neural stem cells can be used to regenerate populations of lost cells in the nervous system.
  • Stem cells are pluripotent cells that self-renew and proliferate in adult life through characteristic asymmetric divisions in which a daughter cell is forced to differentiate and the rest remains as stem cells. There are several types of stem cells depending on the organ in which they are located and their potential differentiation.
  • Ratios can be transformed into heterogeneous cell types by ectopic influences, inducing the generation of a different destination under new environmental signals.
  • mouse bone marrow cells can be incorporated into the central nervous system (CNS) and differentiate into neurons, astrocytes and oligodendrocytes, and that human bone marrow cells can develop a neuronal phenotype in vitro.
  • CNS central nervous system
  • the generation of hematopoietic stem cells and the restoration of blood progenitors from neural stem cells are also known.
  • Neurodegenerative diseases affect populations of neural cells and, in general, the main damage occurs in a specific structural or neurochemical phenotype. In Parkinson's disease, for example, the lost cell type They are mostly dopaminergic neurons of the middle brain.
  • MS Multiple sclerosis
  • neural stem cells are obtained from brain tissue, by tissue biopsy or necropsy, which raises ethical problems and immunological compatibility. Therefore, there is still a need to provide a source of neural stem cells that overcomes the aforementioned drawbacks.
  • the present invention faces the problem of providing a source of neural stem cells.
  • the solution provided by this invention is based on the fact that the inventors have observed that, from hematopoietic stem cells, neural stem cells can be generated both in vitro and in vivo (Examples 2 and 3).
  • Hematopoietic stem cells after administration to a postnatal mammalian animal, can produce neural stem cells that can differentiate into neurons, astrocytes and oligodendrocytes
  • hematopoietic stem cells can be used to produce neural stem cells that, in turn, they are useful for treating neurodegenerative diseases.
  • an object of this invention is the use of hematopoietic stem cells to generate neural stem cells.
  • a further object of this invention is a process for the production of neural stem cells from hematopoietic stem cells.
  • Another additional object of this invention is a pharmaceutical composition comprising hematopoietic stem cells.
  • Another additional object of this invention is the use of hematopoietic stem cells in the preparation of a pharmaceutical composition for the treatment of neurodegenerative diseases.
  • the invention relates, in general, to the use of hematopoietic cells to generate in vivo or in vivo neural stem cells.
  • the invention provides a method for the production of neural stem cells comprising culturing hematopoietic stem cells under conditions that promote the growth and differentiation of said hematopoietic stem cells into neural stem cells.
  • Hematopoietic stem cells can be easily obtained from bone marrow or peripheral blood of an adult mammal (animal or human that has performed all normal development processes) or from the umbilical cord (from mammals including man), by conventional methods.
  • the selection of hematopoietic stem cells from blood extractions or spinal aspirates is done by conventional methods, by for example, by flow cytometry using the appropriate markers for each cell type.
  • Example 1 describes the collection, separation and selection of hematopoietic stem cells from bone marrow and peripheral blood of an adult mammal and from human umbilical cord.
  • Hematopoietic stem cells can be cultured both in vitro and in vivo to obtain neural stem cells.
  • hematopoietic stem cells For in vitro culture of hematopoietic stem cells, said cells are seeded in a suitable culture medium, for example, DMEM (Sigma) or RPMI 1640 (Gibco), and incubated under conditions that allow cell growth. After a few days, usually at 5-6 days, cells that show specific markers of neural stem cells (nestin, radial glia markers and glia acid fibrillar protein [GFAP]) are observed. These results show the in vitro transdifferentiation of hematopoietic stem cells to neural stem cells. In Example 2 a particular embodiment of the in vitro culture of hematopoietic stem cells to obtain neural stem cells is described.
  • DMEM Sigma
  • RPMI 1640 Gibco
  • a physiologically acceptable composition comprising hematopoietic stem cells is administered to a postnatal mammalian animal.
  • the administration of said hematopoietic stem cells can be performed by any suitable method.
  • the administration of said cells to the animal is carried out either by intracerebral injection or by intraparenteral injection (intravenous or intraperitoneal).
  • intracerebral injection or by intraparenteral injection (intravenous or intraperitoneal).
  • Example 3 a particular way of culturing hematopoietic stem cells in order to obtain neural stem cells is described.
  • neural stem cells can be isolated, expanded and used as a source for brain transplants and, therefore, can be used to treat neurodegenerative diseases.
  • the possibility of using hematopoietic stem cells for the treatment of neurodegenerative diseases was tested, obtaining satisfactory and promising results.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of hematopoietic stem cells and a pharmaceutically acceptable carrier.
  • Said pharmaceutical composition may be prepared in any pharmaceutical form of appropriate administration.
  • said pharmaceutical composition is in the form of an injectable intended for intracerebral administration, for example, intraparenchymal or intraventricular, or intraparenteral, for example, intravenous or intraperitoneal.
  • the present invention also relates to the use of hematopoietic stem cells in the preparation of a pharmaceutical composition for the treatment of neurodegenerative diseases and / or pathological situations in which degenerated neuronal cells exist, after surgical or traumatic losses, both in localized processes and in multifocal
  • neural diseases includes all kinds of pathologies, alterations or situations where degenerative loss of neural cells occurs.
  • the therapeutic activity of hematopoietic stem cells in the treatment of neurodegenerative diseases derives from their ability to differentiate in vivo in neural stem cells, which, in turn, can migrate to the region that suffers from the neurodegenerative lesion, invade the injured region and generate the specific population of appropriate neuronal cells.
  • the inventors have conducted numerous studies on the effect of the administration of physiologically acceptable compositions containing hematopoietic stem cells in brains of normal and injured animals.
  • the administration of said compositions is carried out by intracerebral injection, for example, intra-parenchymal or intraventricular, or by intraparenteral injection, for example, intravenous or intraperitoneal.
  • Example 4 describes in vivo experiments that demonstrate the efficacy of the administration of hematopoietic stem cells both intracerebrally and intraparenterally, in the regeneration of lost neuronal populations, and, consequently, their potential usefulness in the treatment of neurodegenerative diseases that occur with the loss of neuronal populations.
  • hematopoietic stem cells can be used to generate neural stem cells, which in turn can be used to regenerate degenerated neural cells or neural losses, after surgical interventions or traumatic, both in localized and multifocal processes.
  • said hematopoietic stem cells can be used both in cell therapy of neurodegenerative diseases, for example, multiple sclerosis (regeneration of oligodendrocytes), amyotrophic lateral sclerosis (regeneration of motor marrow neurons), Parkinson's disease ( regeneration of dopaminergic neurons), Alzheimer's disease (regeneration of cholinergic neurons and hippocampal neurons), degenerative olivócerebellar ataxias (regeneration of olive neurons and Purkinje cells), human spongiform encephalitis or Creutzfeld-Jacob disease, etc., as in therapy replacement and / or regenerative in post-surgical and post-traumatic processes.
  • neurodegenerative diseases for example, multiple sclerosis (regeneration of oligodendrocytes), amyotrophic
  • Hematopoietic stem cells can be administered in any pharmaceutically acceptable form of administration.
  • said hematopoietic stem cells are formulated in the form of injectables intended for administration intracerebrally, for example, intraparenchymal or intraventricular, or intraparenteral, for example, intravenous or intraperitoneal.
  • the amount and manner of administering hematopoietic stem cells to a patient suffering from a neurodegenerative disease in need of treatment should be determined by the doctor who will set the pattern and how to proceed in view of, among other factors, the severity of the disease. and the general condition of the patient. In the same way it will proceed for post-traumatic, post-surgical or post-surgical regeneration processes. functional psychosis
  • hematopoietic stem cells as a cellular source to generate both in vitro and in vivo neural stem cells has numerous advantages since, on the one hand, they are the progeniture cells that can be obtained more easily, for example, from peripheral blood of adult or umbilical cord animals, without having to resort to performing biopsies or necropsies to obtain neural stem cells, which allows to overcome, in addition to discomfort for the patient, obvious ethical problems; and, on the other hand, the possibility of transforming hematopoietic stem cells into neural stem cells makes it possible to have a permanent source of neural stem cells from the individual or from compatible donors, avoiding immunogenic compatibility problems.
  • the possibility of obtaining neural stem cells from hematopoietic stem cells will provide a source of neural stem cells that can be used to restore neuronal cells in neurodegenerative processes. Additionally, the teachings provided by this invention carry various effects, for example, by being able to easily obtain neural stem cells in large quantities, studies on the biology of the process of transdifferentiation between hematopoietic to neural stem cells can be advanced significantly; and also the real possibilities of therapeutic t use of stem cells (hematopoietic or neural) thanks to the possibility of obtaining cells expand patient self
  • the possibility of manipulating the stem cells before being used in cell therapy will allow to induce, generate and / or modify the biological parameters that could influence their therapeutic capacity, such as greater resistance to the harmful process (immunological, genetic or metabolic), greater proliferative capacity, directed toxicity, etc.
  • the medullary tissue is aspirated and collected in DMEM (Dubelcco's modified Eagle medium, Sigma) supplemented with 10% fetal bovine serum (SFB), at 41C. It is mechanically dissociated by repeatedly pipetting under sterile conditions, carefully, it is centrifuged at 1,200 rpm for 6 minutes and resuspended in 0.144M ammonium chloride dissolved in 17 mM Tris buffer, pH 7.2, for 5 minutes. It is then washed with DMEM-SFB and resuspended in the same medium in defined volume for counting and cytometry selection.
  • DMEM Dubelcco's modified Eagle medium, Sigma
  • FFB fetal bovine serum
  • said hematopoietic stem cells are separated and selected by flow cytometry using the appropriate markers depending on the origin of the hematopoietic stem cells to be separated, for example, by using the following markers: a) for mouse cells: markers CD117 (BD Pharmingen) and Sca-1 (BD Pharmingen) for cytometry or, alternatively, selection by size once the cytometer has been calibrated for the CD117 and Sca-1 positive population; and b) for human cells: CD34 markers (BD Pharmingen).
  • hematopoietic stem cells can be obtained from peripheral blood after mobilization of the medullary precursors with granulocyte stimulating factor (G-CSF). Peripheral blood is extracted and hematopoietic stem cells are directly selected, for example, by the use of specific fluorescent markers. The cellular media and the protocol to be followed for obtaining, separating and selecting hematopoietic stem cells are the same as those described in Example 1.1.
  • G-CSF granulocyte stimulating factor
  • Hematopoietic stem cells can be obtained from umbilical cord. For this, umbilical cord bleeding is performed and the parents are selected, for example, by cytometry using appropriate markers. The cellular media and the protocol to be followed for obtaining, separating and selecting hematopoietic stem cells are the same as those described in Example 1.1.
  • a physiologically acceptable composition containing hematopoietic mammalian stem cells is administered to a neonatal mouse, such as a cell suspension in DMEM-SFB enriched in hematopoietic stem cells obtained by the procedure described in Example 1 (in any of its alternatives).
  • hematopoietic stem cells in the animal is well done by intracerebral injection (stereotactic injection of 200,000-300,000 cells dissolved in 1 microliter of RPMI 1640 medium (Gibco) or DMEM (Sigma) enriched with 10% SFB, in brain parenchyma or lateral ventricle) or 2,000,000- 3,000,000 cells in 100 microliters of medium (RPMI 1640) by intraparenteral injection (intravenous or intraperitoneal).
  • hematopoietic stem cell injection At 4-6 days after hematopoietic stem cell injection, the presence of cells showing specific markers of neural stem cells is observed in the recipient animal: nestin, radial glia markers and GFAP (determined as indicated in Example 2) , which shows the transdifferentiation in vivo from hematopoietic stem cells to neural stem cells.
  • a cell suspension of said transplanted brain can be obtained and cultured said cells under appropriate conditions to select neural stem cells [DMEM, 10% SFB, 20 ng / ml of Bfgf (Fibriblest growth basic factor), 20 ng / ml of EGF (Epidermal growth factor) and 1,000 U / ml of LIF (leukemia inhibitor factor)].
  • DMEM neural stem cells
  • SFB 10% SFB
  • Bfgf Fibriblest growth basic factor
  • EGF Epidermal growth factor
  • LIF leukemia inhibitor factor
  • hematopoietic adult mammalian stem cells can become neural stem cells, which, in addition to presenting specific molecular characteristics of this cell type, behave biologically as such, generating neural cells in vivo.
  • oligodendrocyte progenitors (04+) from the donor are observed that accumulate peripherally and invade the demyelinated region.
  • hematopoietic stem cells injected into the brains of adult mammals with demyelinating lesions can migrate attracted by the injured region, invade the lesion and generate oligodendroglial line cells.
  • a composition comprising a suspension of hematopoietic stem cells is injected into a physiologically acceptable vehicle (2,000,000 cells in 100 microliths of RPMI 1640 solution) intravenously or intraperitoneally (bone marrow cells) according to Example 1

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Abstract

The invention relates to the production of neural stem cells consisting in cultivating haematopoietic stem cells in conditions that promote the growth and differentiation of said haematopoietic stem cells into neural stem cells which can be used to regenerate populations of cells lost in the nervous system. The invention also relates to the use of haematopoietic stem cells in the production of a pharmaceutical composition that is used to treat neurodegenerative diseases and/or pathological conditions involving degenerated neuronal cells, both in localised and multifocal processes.

Description

EMPLEO DE CÉLULAS MADRE HEMATOPOYETICAS EN LA GENERACIÓN DEEMPLOYMENT OF HEMATOPOYETIC MOTHER CELLS IN THE GENERATION OF
CÉLULAS MADRE NEURALESNEURAL MOTHER CELLS
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La invención se refiere a la generación de células madre neurales, tanto in vi tro como in vivo, a partir de células madre hematopoyéticas. Las células madre neurales pueden usarse para regenerar poblaciones de células perdidas en el sistema nervioso.The invention relates to the generation of neural stem cells, both in vitro and in vivo, from hematopoietic stem cells. Neural stem cells can be used to regenerate populations of lost cells in the nervous system.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Las células madre son células pluripotentes que se auto- renuevan y que proliferan en la vida adulta mediante divisiones asimétricas características en las que una célula hija se ve forzada a diferenciarse y el resto permanece como células madre. Existen diversos tipos de células madre dependiendo del órgano en el que estén localizadas y de su diferenciación potencial .Stem cells are pluripotent cells that self-renew and proliferate in adult life through characteristic asymmetric divisions in which a daughter cell is forced to differentiate and the rest remains as stem cells. There are several types of stem cells depending on the organ in which they are located and their potential differentiation.
Las células madre de adulto pueden ser transformadas en tipos heterogénicos de células mediante influencias ectópicas, induciendo la generación de un destino diferente bajo nuevas señales ambientales. En este sentido, se ha descrito que células de médula ósea de ratón pueden ser incorporadas en el sistema nervioso central (SNC) y diferenciarse en neuronas, astrocitos y oligodendrocitos, y que células de médula ósea humana pueden desarrollar un fenotipo neuronal in vi tro . También se conoce la generación de células madre hematopoyéticas y la restauración de progenitores sanguíneos, a partir de células madre neurales. Las enfermedades neurodegenerativas afectan a poblaciones de células neurales y, en general, el daño principal ocurre en un fenotipo estructural o neuroqui ico especifico. En la enfermedad de Parkinson, por ejemplo, el tipo celular perdido mayoritariamente son neuronas dopaminérgicas del cerebro medio. La esclerosis múltiple (EM) es una enfermedad autoinmune inflamatoria del SNC caracterizada por la existencia de placas desmielinizadas diseminadas que evolucionan progresivamente hacia lesiones crónicas. En el caso de la EM existen evidencias de que en los estadios iniciales de la enfermedad, los progenitores de oligodendrocitos se activan para reactivar el proceso de remielinización. Cuando este intento regenerativo no tiene éxito, la placa desmielinizada se convierte en una lesión crónica.Adult stem cells can be transformed into heterogeneous cell types by ectopic influences, inducing the generation of a different destination under new environmental signals. In this sense, it has been described that mouse bone marrow cells can be incorporated into the central nervous system (CNS) and differentiate into neurons, astrocytes and oligodendrocytes, and that human bone marrow cells can develop a neuronal phenotype in vitro. The generation of hematopoietic stem cells and the restoration of blood progenitors from neural stem cells are also known. Neurodegenerative diseases affect populations of neural cells and, in general, the main damage occurs in a specific structural or neurochemical phenotype. In Parkinson's disease, for example, the lost cell type They are mostly dopaminergic neurons of the middle brain. Multiple sclerosis (MS) is an inflammatory autoimmune CNS disease characterized by the existence of disseminated demyelinated plaques that progressively progress towards chronic lesions. In the case of MS there is evidence that in the initial stages of the disease, oligodendrocyte progenitors are activated to reactivate the remyelination process. When this regenerative attempt is unsuccessful, the demyelinated plaque becomes a chronic lesion.
El reemplazamiento funcional de poblaciones neuronales especificas mediante transplante de tejido neural representa una estrategia terapéutica atractiva para tratar enfermedades neurodegenerativas . Sin embargo, actualmente, las células madre neurales se obtienen a partir de tejido cerebral, mediante biopsia de tejido o necropsia, lo que plantea problemas éticos y de compatibilidad inmunológica. Por tanto, sigue existiendo la necesidad de proporcionar una fuente de células madre neurales que supere los inconvenientes mencionados.Functional replacement of specific neuronal populations by neural tissue transplantation represents an attractive therapeutic strategy to treat neurodegenerative diseases. However, currently, neural stem cells are obtained from brain tissue, by tissue biopsy or necropsy, which raises ethical problems and immunological compatibility. Therefore, there is still a need to provide a source of neural stem cells that overcomes the aforementioned drawbacks.
COMPENDIO DE LA INVENCIÓNSUMMARY OF THE INVENTION
La presente invención se enfrenta con el problema de proporcionar una fuente de células madre neurales. La solución proporcionada por esta invención se basa en que los inventores han observado que, a partir de células madre hematopoyéticas se pueden generar células madre neurales tanto in vi tro como in vivo (Ejemplos 2 y 3) . Las células madre hematopoyéticas, tras su administración a un animal mamífero postnatal, pueden producir células madre neurales que pueden diferenciarse en neuronas, astrocitos y oligodendrocitosThe present invention faces the problem of providing a source of neural stem cells. The solution provided by this invention is based on the fact that the inventors have observed that, from hematopoietic stem cells, neural stem cells can be generated both in vitro and in vivo (Examples 2 and 3). Hematopoietic stem cells, after administration to a postnatal mammalian animal, can produce neural stem cells that can differentiate into neurons, astrocytes and oligodendrocytes
(Ejemplo 4). Por tanto, las células madre hematopoyéticas pueden ser utilizadas para producir células madre neurales que, a su vez, son útiles para tratar enfermedades neurodegenerativas .(Example 4). Therefore, hematopoietic stem cells can be used to produce neural stem cells that, in turn, they are useful for treating neurodegenerative diseases.
Por tanto, un objeto de esta invención lo constituye el empleo de células madre hematopoyéticas para generar células madre neurales.Therefore, an object of this invention is the use of hematopoietic stem cells to generate neural stem cells.
Un objeto adicional de esta invención lo constituye un procedimiento para la producción de células madre neurales a partir de células madre hematopoyéticas.A further object of this invention is a process for the production of neural stem cells from hematopoietic stem cells.
Otro objeto adicional de esta invención lo constituye una composición farmacéutica que comprende células madre hematopoyéticas .Another additional object of this invention is a pharmaceutical composition comprising hematopoietic stem cells.
Otro objeto adicional de esta invención lo constituye el empleo de células madre hematopoyéticas en la elaboración de una composición farmacéutica para el tratamiento de enfermedades neurodegenerativas.Another additional object of this invention is the use of hematopoietic stem cells in the preparation of a pharmaceutical composition for the treatment of neurodegenerative diseases.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
La invención se refiere, en general, al empleo de células hematopoyéticas para generar in vi tro o in vivo células madre neurales .The invention relates, in general, to the use of hematopoietic cells to generate in vivo or in vivo neural stem cells.
En un aspecto, la invención proporciona un procedimiento para la producción de células madre neurales que comprende cultivar células madre hematopoyéticas bajo condiciones que promueven el crecimiento y la diferenciación de dichas células madre hematopoyéticas en células madre neurales.In one aspect, the invention provides a method for the production of neural stem cells comprising culturing hematopoietic stem cells under conditions that promote the growth and differentiation of said hematopoietic stem cells into neural stem cells.
Las células madre hematopoyéticas pueden obtenerse fácilmente a partir de médula ósea o sangre periférica de un mamífero adulto (animal o humano que ha realizado todos los procesos normales de desarrollo) o a partir del cordón umbilical (de mamíferos incluido el hombre) , mediante métodos convencionales. La selección de las células madre hematopoyéticas a partir de las extracciones sanguíneas o aspirado medular se realiza por métodos convencionales, por ejemplo, por citometría de flujo utilizando los marcadores apropiados para cada tipo celular. En el Ejemplo 1 se describe la obtención, separación y selección de células madre hematopoyéticas a partir de médula ósea y sangre periférica de un mamífero adulto y a partir de cordón umbilical humano.Hematopoietic stem cells can be easily obtained from bone marrow or peripheral blood of an adult mammal (animal or human that has performed all normal development processes) or from the umbilical cord (from mammals including man), by conventional methods. The selection of hematopoietic stem cells from blood extractions or spinal aspirates is done by conventional methods, by for example, by flow cytometry using the appropriate markers for each cell type. Example 1 describes the collection, separation and selection of hematopoietic stem cells from bone marrow and peripheral blood of an adult mammal and from human umbilical cord.
Las células madre hematopoyéticas pueden ser cultivadas tanto in vi tro como in vivo para obtener células madre neurales .Hematopoietic stem cells can be cultured both in vitro and in vivo to obtain neural stem cells.
Para el cultivo in vi tro de células madre hematopoyéticas se siembran dichas células en un medio de cultivo adecuado, por ejemplo, DMEM (Sigma) o RPMI 1640 (Gibco) , y se incuban bajo condiciones que permiten el crecimiento celular. Al cabo de unos pocos días, normalmente a los 5-6 días, se observan células que manifiestan marcadores específicos de células madre neurales (nestina, marcadores de glía radial y proteína fibrilar acida de la glia [GFAP] ) . Estos resultados ponen de manifiesto la transdiferenciación in vi tro de células madre hematopoyéticas a células madre neurales. En el Ejemplo 2 se describe una realización particular del cultivo in vi tro de células madre hematopoyéticas para obtener células madre neurales .For in vitro culture of hematopoietic stem cells, said cells are seeded in a suitable culture medium, for example, DMEM (Sigma) or RPMI 1640 (Gibco), and incubated under conditions that allow cell growth. After a few days, usually at 5-6 days, cells that show specific markers of neural stem cells (nestin, radial glia markers and glia acid fibrillar protein [GFAP]) are observed. These results show the in vitro transdifferentiation of hematopoietic stem cells to neural stem cells. In Example 2 a particular embodiment of the in vitro culture of hematopoietic stem cells to obtain neural stem cells is described.
Para el cultivo in vivo de células madre hematopoyéticas se administra a un animal mamífero postnatal una composición fisiológicamente aceptable que comprende células madre hematopoyéticas. La administración de dichas células madre hematopoyéticas se puede realizar por cualquier método adecuado. En una realización particular, la administración de dichas células al animal se realiza bien mediante inyección intracerebral o bien mediante inyección intraparenteral (intravenosa o intraperitoneal) . Al cabo de 4-6 días postinyección se observa en el mamífero receptor la presencia de células que manifiestan marcadores específicos de células madre neurales (nestina, marcadores de glía radial y GFAP) , lo que pone de manifiesto la transdiferenciación in vivo desde células madre hematopoyéticas a células madre neurales. En el Ejemplo 3 se describe una forma particular de cultivar in vi tro células madre hematopoyéticas para obtener células madre neurales. Como es conocido, las células madre neurales pueden ser aisladas, expandidas y utilizadas como fuente para transplantes cerebrales y, por tanto, pueden ser utilizadas para tratar enfermedades neurodegenerativas. A la vista de los resultados obtenidos, en particular, de la generación in vivo de células madre neurales a partir de células madre hematopoyéticas, se ensayó la posibilidad de utilizar células madre hematopoyéticas para el tratamiento de enfermedades neurodegenerativas, obteniéndose unos resultados satisfactorios y esperanzadores .For the in vivo culture of hematopoietic stem cells, a physiologically acceptable composition comprising hematopoietic stem cells is administered to a postnatal mammalian animal. The administration of said hematopoietic stem cells can be performed by any suitable method. In a particular embodiment, the administration of said cells to the animal is carried out either by intracerebral injection or by intraparenteral injection (intravenous or intraperitoneal). After 4-6 days post-injection, the presence of cells manifesting specific markers of neural stem cells (nestin, radial glia markers and GFAP) is observed in the recipient mammal, which highlights in vivo transdifferentiation from hematopoietic stem cells to neural stem cells. In Example 3 a particular way of culturing hematopoietic stem cells in order to obtain neural stem cells is described. As is known, neural stem cells can be isolated, expanded and used as a source for brain transplants and, therefore, can be used to treat neurodegenerative diseases. In view of the results obtained, in particular, of the in vivo generation of neural stem cells from hematopoietic stem cells, the possibility of using hematopoietic stem cells for the treatment of neurodegenerative diseases was tested, obtaining satisfactory and promising results.
La invención también proporciona una composición farmacéutica que comprende una cantidad terapéuticamente eficaz de células madre hematopoyéticas y un vehículo farmacéuticamente aceptable. Dicha composición farmacéutica puede prepararse en cualquier forma farmacéutica de administración apropiada. En una realización particular, dicha composición farmacéutica se presenta en forma de un inyectable destinado a su administración por vía intracerebral, por ejemplo, intraparenquimatosa o intraventricular, o intraparenteral, por ejemplo, intravenosa o intraperitoneal. Una revisión general de las distintas formas farmacéuticas de administración de productos con actividad terapéutica puede encontrarse, por ejemplo, en el Tratado de Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A. de Ediciones, (1993).The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of hematopoietic stem cells and a pharmaceutically acceptable carrier. Said pharmaceutical composition may be prepared in any pharmaceutical form of appropriate administration. In a particular embodiment, said pharmaceutical composition is in the form of an injectable intended for intracerebral administration, for example, intraparenchymal or intraventricular, or intraparenteral, for example, intravenous or intraperitoneal. A general review of the different pharmaceutical forms of administration of products with therapeutic activity can be found, for example, in the Treaty of Farmacia Galenica, C. Faulí i Trillo, Luzán 5, S.A. of Editions, (1993).
La presente invención también se refiere al empleo de células madre hematopoyéticas en la elaboración de una composición farmacéutica para el tratamiento de enfermedades neurodegenerativas y/o de situaciones patológicas en las que existen células neuronales degeneradas, tras pérdidas quirúrgicas o traumáticas, tanto en procesos localizados como multifocales .The present invention also relates to the use of hematopoietic stem cells in the preparation of a pharmaceutical composition for the treatment of neurodegenerative diseases and / or pathological situations in which degenerated neuronal cells exist, after surgical or traumatic losses, both in localized processes and in multifocal
La expresión "enfermedades neurodegenerativas", tal como se utiliza en esta descripción, incluye todo tipo de patologías, alteraciones o situaciones en donde se produce la pérdida degenerativa de células neurales.The expression "neurodegenerative diseases", as used in this description, includes all kinds of pathologies, alterations or situations where degenerative loss of neural cells occurs.
La actividad terapéutica de las células madre hematopoyéticas en el tratamiento de las enfermedades neurodegenerativas deriva de su capacidad de diferenciarse in vivo en células madre neurales, las cuales, a su vez, pueden migrar hacia la región que padece la lesión neurodegenerativa, invadir la región lesionada y generar la población específica de células neuronales apropiadas.The therapeutic activity of hematopoietic stem cells in the treatment of neurodegenerative diseases derives from their ability to differentiate in vivo in neural stem cells, which, in turn, can migrate to the region that suffers from the neurodegenerative lesion, invade the injured region and generate the specific population of appropriate neuronal cells.
Para alcanzar los resultados proporcionados por esta invención, los inventores han realizado numerosos estudios sobre el efecto de la administración de composiciones fisiológicamente aceptables que contenían células madre hematopoyéticas en cerebros de animales normales y lesionados. En una realización particular, la administración de dichas composiciones se realiza mediante inyección intracerebral, por ejemplo, intra-parenquimatosa o intraventricular, o bien mediante inyección intraparenteral, por ejemplo, intravenosa o intraperitoneal. En el Ejemplo 4 se describen unos experimentos in vivo que ponen de manifiesto la eficacia de la administración de células madre hematopoyéticas tanto por vía intracerebral como por vía intraparenteral, en la regeneración de poblaciones neuronales perdidas, y, consecuentemente, su potencial utilidad en el tratamiento de enfermedades neurodegenerativas que cursen con la pérdida de poblaciones neuronales . Por tanto, se pueden emplear células madre hematopoyéticas para generar células madre neurales, que a su vez pueden ser utilizadas para regenerar células neurales degeneradas o pérdidas neurales, tras intervenciones quirúrgicas o traumáticas, tanto en procesos localizados como multifocales . A modo ilustrativo, no limitativo, dichas células madre hematopoyéticas se pueden utilizar tanto en terapia celular de enfermedades neurodegenerativas, por ejemplo, esclerosis múltiple (regeneración de oligodendrocitos), esclerosis lateral amiotrófica (regeneración de neuronas motoras de la médula) , enfermedad de Parkinson (regeneración de neuronas dopaminérgicas) , enfermedad de Alzheimer (regeneración de neuronas colinérgicas y neuronas del hipocampo) , ataxias olivócerebelosas degenerativas (regeneración de neuronas olivares y células de Purkinje) , encefalitis espongiforme humana o Enfermedad de Creutzfeld-Jacob, etc., como en terapia sustitutiva y/o regenerativa en procesos postquirúrgicos y post-traumáticos. Por otra parte, dado que actualmente se está cada vez más cerca de determinar la base biológica de las psicosis funcionales (esquizofrenia, enfermedad bipolar y síndromes obsesivos) , la terapia celular podría servir para restablecer circuitos no establecidos o restaurar conexiones aberrantes . Las células madre hematopoyéticas pueden administrarse en cualquier forma de administración farmacéuticamente aceptable. En una realización particular, dichas células madre hematopoyéticas se formulan en forma de inyectables destinados a su administración por vía intracerebral, por ejemplo, intra- parenquimatosa o intraventricular, o intraparenteral, por ejemplo, intravenosa o intraperitoneal.To achieve the results provided by this invention, the inventors have conducted numerous studies on the effect of the administration of physiologically acceptable compositions containing hematopoietic stem cells in brains of normal and injured animals. In a particular embodiment, the administration of said compositions is carried out by intracerebral injection, for example, intra-parenchymal or intraventricular, or by intraparenteral injection, for example, intravenous or intraperitoneal. Example 4 describes in vivo experiments that demonstrate the efficacy of the administration of hematopoietic stem cells both intracerebrally and intraparenterally, in the regeneration of lost neuronal populations, and, consequently, their potential usefulness in the treatment of neurodegenerative diseases that occur with the loss of neuronal populations. Therefore, hematopoietic stem cells can be used to generate neural stem cells, which in turn can be used to regenerate degenerated neural cells or neural losses, after surgical interventions or traumatic, both in localized and multifocal processes. By way of illustration, not limitation, said hematopoietic stem cells can be used both in cell therapy of neurodegenerative diseases, for example, multiple sclerosis (regeneration of oligodendrocytes), amyotrophic lateral sclerosis (regeneration of motor marrow neurons), Parkinson's disease ( regeneration of dopaminergic neurons), Alzheimer's disease (regeneration of cholinergic neurons and hippocampal neurons), degenerative olivócerebellar ataxias (regeneration of olive neurons and Purkinje cells), human spongiform encephalitis or Creutzfeld-Jacob disease, etc., as in therapy replacement and / or regenerative in post-surgical and post-traumatic processes. On the other hand, since it is currently getting closer to determining the biological basis of functional psychoses (schizophrenia, bipolar disease and obsessive syndromes), cell therapy could be used to restore non-established circuits or restore aberrant connections. Hematopoietic stem cells can be administered in any pharmaceutically acceptable form of administration. In a particular embodiment, said hematopoietic stem cells are formulated in the form of injectables intended for administration intracerebrally, for example, intraparenchymal or intraventricular, or intraparenteral, for example, intravenous or intraperitoneal.
La cantidad y forma de administrar las células madre hematopoyéticas a un paciente que padece una enfermedad neurodegenerativa en necesidad de tratamiento deberá ser fijada por el facultativo quien fijará la pauta y forma de proceder a la vista de, entre otros factores, la gravedad de la enfermedad y el estado general del paciente. Del mismo modo se procederá para procesos de regeneración postraumática, postquirúrgica o psicosis funcional.The amount and manner of administering hematopoietic stem cells to a patient suffering from a neurodegenerative disease in need of treatment should be determined by the doctor who will set the pattern and how to proceed in view of, among other factors, the severity of the disease. and the general condition of the patient. In the same way it will proceed for post-traumatic, post-surgical or post-surgical regeneration processes. functional psychosis
El empleo de células madre hematopoyéticas como fuente celular para generar tanto in vitro como in vivo células madre neurales presenta numerosas ventajas ya que, por una parte, son las células progenituras que se pueden obtener más fácilmente, por ejemplo, a partir de sangre periférica de animales adultos o de cordón umbilical, sin tener que recurrir a la realización de biopsias ni necropsias para obtener células madre neurales, lo que permite superar, además de molestias para el paciente, evidentes problemas, éticos; y, por otra parte, la posibilidad de transformar células madre hematopoyéticas en células madre neurales permite tener una fuente permanente de células madre neurales del propio individuo o de donantes compatibles evitándose problemas de compatibilidad inmunogénica. La posibilidad de obtener células madre neurales a partir de células madre hematopoyéticas permitirá disponer de una fuente de células madre neurales que podrán ser utilizadas para restaurar células neuronales en procesos neurodegenerativos. Adicionalmente, las enseñanzas proporcionadas por esta invención llevan consigo diversos efectos, por ejemplo, al poder obtenerse fácilmente células madre neurales en cantidades elevadas se podrán avanzar de forma significativa los estudios sobre la biología del proceso de transdiferenciación entre células madre de hematopoyéticas a neurales; y, además, se amplían las posibilidades reales de usot terapéutico de las células madre (hematopoyéticas o neurales) gracias a la posibilidad de obtener células del propio pacienteThe use of hematopoietic stem cells as a cellular source to generate both in vitro and in vivo neural stem cells has numerous advantages since, on the one hand, they are the progeniture cells that can be obtained more easily, for example, from peripheral blood of adult or umbilical cord animals, without having to resort to performing biopsies or necropsies to obtain neural stem cells, which allows to overcome, in addition to discomfort for the patient, obvious ethical problems; and, on the other hand, the possibility of transforming hematopoietic stem cells into neural stem cells makes it possible to have a permanent source of neural stem cells from the individual or from compatible donors, avoiding immunogenic compatibility problems. The possibility of obtaining neural stem cells from hematopoietic stem cells will provide a source of neural stem cells that can be used to restore neuronal cells in neurodegenerative processes. Additionally, the teachings provided by this invention carry various effects, for example, by being able to easily obtain neural stem cells in large quantities, studies on the biology of the process of transdifferentiation between hematopoietic to neural stem cells can be advanced significantly; and also the real possibilities of therapeutic t use of stem cells (hematopoietic or neural) thanks to the possibility of obtaining cells expand patient self
(homotransplante) o de un donante de médula ósea compatible(homotransplant) or from a compatible bone marrow donor
(alotransplante) . Por otra parte, puesto que muchas formas de procesos neurodegenerativos presentan multifocalidad, la única forma de actuar con terapia celular de forma global sobre todas las lesiones es la administración, por ejemplo, sistémica de los elementos terapéuticos. La administración sistémica (intravenosa o intraperitoneal) permite el acceso de las células madre neurales a todos los lugares donde se localiza la lesión. Esto es de fundamental interés en procesos multifocales tales como la EM, la esclerosis lateral amiotrófica (ELA) , etc.(allogeneic transplant). On the other hand, since many forms of neurodegenerative processes have multifocality, the only way to act with global cell therapy on all lesions is the administration, for example, systemic administration of therapeutic elements Systemic administration (intravenous or intraperitoneal) allows the access of neural stem cells to all places where the lesion is located. This is of fundamental interest in multifocal processes such as MS, amyotrophic lateral sclerosis (ALS), etc.
La posibilidad de manipular las células madre antes de ser utilizadas en terapia celular, permitirá inducir, generar y/o modificar los parámetros biológicos que pudieran influir en su capacidad terapéutica, tales como mayor resistencia al proceso lesivo (inmunológico, genético o metabólico) , mayor capacidad proliferativa, toxicidad dirigida, etc.The possibility of manipulating the stem cells before being used in cell therapy, will allow to induce, generate and / or modify the biological parameters that could influence their therapeutic capacity, such as greater resistance to the harmful process (immunological, genetic or metabolic), greater proliferative capacity, directed toxicity, etc.
Los siguientes ejemplos ilustran la invención y no deben ser considerados limitativos del alcance de la misma.The following examples illustrate the invention and should not be considered as limiting its scope.
EJEMPLO 1EXAMPLE 1
Obtención y selección de células madre hematopoyéticasObtaining and selecting hematopoietic stem cells
1.1 A partir de médula ósea de un mamífero adulto (cualquier especie de mamífero que ha superado el periodo neonatal de desarrollo postnatal, incluido el hombre)1.1 From the bone marrow of an adult mammal (any species of mammal that has exceeded the neonatal period of postnatal development, including man)
El tejido medular se aspira y recoge en DMEM (Dubelcco's modified Eagle médium, Sigma) suplementado con 10% de suero fetal bovino (SFB) , a 41C. Se disocia mecánicamente pipeteando repetidamente en condiciones de esterilidad, con cuidado, se centrifuga a 1.200 rpm durante 6 minutos y resuspende en cloruro amónico 0,144 M disuelto en tampón Tris 17 mM, pH 7,2, durante 5 minutos. A continuación, se lava con DMEM-SFB y se resuspende en el mismo medio en volumen definido para contaje y selección por citometría. A partir de la suspensión celular obtenida que contiene las células madre hematopoyéticas se procede a la separación y selección de dichas células madre hematopoyéticas mediante citometría de flujo utilizando los marcadores apropiados dependiendo del origen de las células madre hematopoyéticas a separar, por ejemplo, mediante el empleo de los siguientes marcadores : a) para células de ratón: marcadores CD117 (BD Pharmingen) y Sca-1 (BD Pharmingen) para citometría o, alternativamente, selección por tamaño una vez calibrado el citómetro para la población CD117 y Sca-1 positiva; y b) para células humanas: marcadores CD34 (BD Pharmingen).The medullary tissue is aspirated and collected in DMEM (Dubelcco's modified Eagle medium, Sigma) supplemented with 10% fetal bovine serum (SFB), at 41C. It is mechanically dissociated by repeatedly pipetting under sterile conditions, carefully, it is centrifuged at 1,200 rpm for 6 minutes and resuspended in 0.144M ammonium chloride dissolved in 17 mM Tris buffer, pH 7.2, for 5 minutes. It is then washed with DMEM-SFB and resuspended in the same medium in defined volume for counting and cytometry selection. From the obtained cell suspension containing the hematopoietic stem cells, said hematopoietic stem cells are separated and selected by flow cytometry using the appropriate markers depending on the origin of the hematopoietic stem cells to be separated, for example, by using the following markers: a) for mouse cells: markers CD117 (BD Pharmingen) and Sca-1 (BD Pharmingen) for cytometry or, alternatively, selection by size once the cytometer has been calibrated for the CD117 and Sca-1 positive population; and b) for human cells: CD34 markers (BD Pharmingen).
1.2 A partir de sangre periférica de un mamífero adulto (cualquier especie de mamífero que ha superado el periodo neonatal de desarrollo postnatal, incluido el hombre)1.2 From peripheral blood of an adult mammal (any species of mammal that has exceeded the neonatal period of postnatal development, including man)
Células madre hematopoyéticas de mamífero adulto pueden obtenerse a partir de sangre periférica tras movilización de los precursores medulares con factor estimulador de los granulocitos (G-CSF) . La sangre periférica se extrae y las células madre hematopoyéticas se seleccionan directamente, por ejemplo, mediante el empleo de marcadores fluorescentes específicos. Los medios celulares y el protocolo a seguir para la obtención, separación y selección de células madre hematopoyéticas son los mismos que los descritos en el Ejemplo 1.1.Adult mammalian hematopoietic stem cells can be obtained from peripheral blood after mobilization of the medullary precursors with granulocyte stimulating factor (G-CSF). Peripheral blood is extracted and hematopoietic stem cells are directly selected, for example, by the use of specific fluorescent markers. The cellular media and the protocol to be followed for obtaining, separating and selecting hematopoietic stem cells are the same as those described in Example 1.1.
1.3 A partir de cordón umbilical humano Células madre hematopoyéticas pueden obtenerse a partir de cordón umbilical. Para ello, se realiza un sangrado del cordón umbilical y se seleccionan los progenitores, por ejemplo, mediante citometría utilizando marcadores apropiados. Los medios celulares y el protocolo a seguir para la obtención, separación y selección de células madre hematopoyéticas son los mismos que los descritos en el Ejemplo 1.1.1.3 From human umbilical cord Hematopoietic stem cells can be obtained from umbilical cord. For this, umbilical cord bleeding is performed and the parents are selected, for example, by cytometry using appropriate markers. The cellular media and the protocol to be followed for obtaining, separating and selecting hematopoietic stem cells are the same as those described in Example 1.1.
EJEMPLO 2 Cultivo in vitro de células madre hematopoyéticasEXAMPLE 2 In vitro culture of hematopoietic stem cells
Se siembran en placas de 96 pocilios unas 200.000 células por pocilio de células madre hematopoyéticas de mamífero procedentes de una suspensión celular en DMEM-SFB enriquecida en dichas células, obtenida mediante el procedimiento descrito en el Ejemplo 1 (en cualquiera de sus alternativas) . Al cabo de 5 ó 6 días en cultivo (a 37°C, con un 10%de C02) en condiciones que permiten el crecimiento celular se observan células con características, determinadas por la manifestación de marcadores específicos, de células madre neurales, tales como: nestina: determinado por reacción con un anticuerpo monoclonal anti-nestina (Chemicon) ; marcadores de glía radial: determinados con el anticuerpo 40E-C (Hybrodoma Banck) , y GFAP: determinado con anticuerpos policlonales (Chemicon).About 200,000 cells are seeded in 96-well plates per well of mammalian hematopoietic stem cells from a cell suspension in DMEM-SFB enriched in said cells, obtained by the procedure described in Example 1 (in any of its alternatives). After 5 or 6 days in culture (at 37 ° C, with 10% C0 2 ) under conditions that allow cell growth, cells with characteristics, determined by the manifestation of specific markers, of neural stem cells are observed, such as: nestin: determined by reaction with a monoclonal anti-nestin antibody (Chemicon); Radial glia markers: determined with the 40E-C antibody (Hybrodoma Banck), and GFAP: determined with polyclonal antibodies (Chemicon).
Estos resultados ponen de manifiesto la transdiferenciación in vitro de células madre hematopoyéticas a células madre neurales.These results show the in vitro transdifferentiation of hematopoietic stem cells to neural stem cells.
EJEMPLO 3EXAMPLE 3
Cultivo in vivo de células madre hematopoyéticasIn vivo culture of hematopoietic stem cells
Para el cultivo in vivo de células madre hematopoyéticas se administra a un ratón neonatal una composición fisiológicamente aceptable que contiene células madre hematopoyéticas de mamífero, tal como una suspensión celular en DMEM-SFB enriquecida en células madre hematopoyéticas obtenida mediante el procedimiento descrito en el Ejemplo 1 (en cualquiera de sus alternativas) . La administración de dichas células madre hematopoyéticas en el animal se realiza bien mediante inyección intracerebral (inyección estereotáxica de 200.000-300.000 células disueltas en 1 microlitro de medio RPMI 1640 (Gibco) o DMEM (Sigma) enriquecidos con 10% de SFB, en parénquima cerebral o ventrículo lateral) o bien 2.000.000- 3.000.000 células en 100 microlitros de medio (RPMI 1640) mediante inyección intraparenteral (intravenosa o intraperitoneal) . A los 4-6 días de la inyección de células madre hematopoyéticas se observa en el animal receptor la presencia de células que manifiestan marcadores específicos de células madre neurales: nestina, marcadores de glía radial y GFAP (determinados como se indica en el Ejemplo 2), lo que pone de manifiesto la transdiferenciación in vivo desde células madre hematopoyéticas a células madre neurales.For the in vivo culture of hematopoietic stem cells, a physiologically acceptable composition containing hematopoietic mammalian stem cells is administered to a neonatal mouse, such as a cell suspension in DMEM-SFB enriched in hematopoietic stem cells obtained by the procedure described in Example 1 (in any of its alternatives). The administration of said hematopoietic stem cells in the animal is well done by intracerebral injection (stereotactic injection of 200,000-300,000 cells dissolved in 1 microliter of RPMI 1640 medium (Gibco) or DMEM (Sigma) enriched with 10% SFB, in brain parenchyma or lateral ventricle) or 2,000,000- 3,000,000 cells in 100 microliters of medium (RPMI 1640) by intraparenteral injection (intravenous or intraperitoneal). At 4-6 days after hematopoietic stem cell injection, the presence of cells showing specific markers of neural stem cells is observed in the recipient animal: nestin, radial glia markers and GFAP (determined as indicated in Example 2) , which shows the transdifferentiation in vivo from hematopoietic stem cells to neural stem cells.
EJEMPLO 4 Experimentos in vivoEXAMPLE 4 Experiments in vivo
4.1 Inyecciones en cerebros de animales normales neonatales Una suspensión celular de 300.000 células en 1 μl de células madre hematopoyéticas obtenidas según el Ejemplo 14.1 Injections in brains of normal neonatal animals A cell suspension of 300,000 cells in 1 μl of hematopoietic stem cells obtained according to Example 1
[1.1] a partir de médula ósea, en un vehículo fisiológicamente aceptable, se inyecta en el cerebro de ratones neonatales (P0) mediante inyección intraparenquimatosa o intraventricular. Al cabo de 4- 6 días post-inyección, se observan células del donante en la región ventricular y subventricular del cerebro del huésped. Estas células presentan características moleculares de células madre neurales: nestina, vimentina y GFAP positivas. A partir del cerebro del animal inyectado (cerebro transplantado) , a los 6 días post-inyección, se puede obtener una suspensión celular de dicho cerebro transplantado y cultivar dichas células en condiciones apropiadas para seleccionar células madre neurales [DMEM, 10% de SFB, 20 ng/ml de Bfgf (Fibriblest growth factor básico) , 20 ng/ml de EGF (Epidermal growth factor) y 1.000 U/ml de LIF (factor inhibidor de leucemia) ] . Al cabo de 5-6 días se observa la formación de neuroesferas y se puede constatar la existencia de abundantes células procedentes del donante. Además, si se inyectan estas células en las mismas condiciones, en cerebro de mamíferos neonatos (ratones P0) o en cerebro lesionado de ratones adultos (véase el Ejemplo 4.2), se consigue activar su proliferación y la producción de células madre neurales con tipologías y características similares a las obtenidas tras la inyección de células madre hematopoyéticas. Estos resultados ponen de manifiesto que las células madre hematopoyéticas de mamífero adulto pueden convertirse en células madre neurales, que, además de presentar características moleculares específicas de este tipo celular, se comportan biológicamente como tales, generando células neurales in vivo .[1.1] from bone marrow, in a physiologically acceptable vehicle, it is injected into the brain of neonatal mice (P0) by intraparenchymal or intraventricular injection. After 4-6 days post-injection, donor cells are observed in the ventricular and subventricular region of the host brain. These cells have molecular characteristics of neural stem cells: nestin, vimentin and GFAP positive. From the brain of the injected animal (transplanted brain), at 6 days post-injection, a cell suspension of said transplanted brain can be obtained and cultured said cells under appropriate conditions to select neural stem cells [DMEM, 10% SFB, 20 ng / ml of Bfgf (Fibriblest growth basic factor), 20 ng / ml of EGF (Epidermal growth factor) and 1,000 U / ml of LIF (leukemia inhibitor factor)]. After 5-6 days the formation of neurospheres is observed and the existence of abundant can be verified cells from the donor. In addition, if these cells are injected under the same conditions, in the brain of newborn mammals (P0 mice) or in the injured brain of adult mice (see Example 4.2), it is possible to activate their proliferation and the production of neural stem cells with typologies and characteristics similar to those obtained after hematopoietic stem cell injection. These results show that hematopoietic adult mammalian stem cells can become neural stem cells, which, in addition to presenting specific molecular characteristics of this cell type, behave biologically as such, generating neural cells in vivo.
Experimentos control, con concentrado celular pero sin células madre hematopoyéticas, es decir, CD117 y Sca-1 negativas, no han mostrado ningún tipo de células neurales procedentes del donante.Control experiments, with cell concentrate but without hematopoietic stem cells, that is, negative CD117 and Sca-1, have not shown any type of neural cells from the donor.
4.2 Inyecciones en cerebros lesionados de animales adultos Para realizar este ensayo se provocan lesiones desmielinizantes en ratones localizadas en el hemisferio cerebral mediante la inyección estereotáxica de lisolecitina. Para ello, mediante un trépano estereotáxico se inocula, lentamente y con una jeringuilla Hamilton, 1 μl de lisolecitina (Sigma) en el parénquima cerebral del telencéfalo. Se intenta localizar la región lateral de la sustancia blanca hemisférica de un lado del cerebro. El estudio anatomo-patológico muestra que en una semana se ha producido una importante desmielinización con abundante proliferación astroglial en la región de la inoculación. Tras localizar la lesión se inyecta una composición que comprende una suspensión de células madre hematopoyéticas en un vehículo fisiológicamente aceptable, en el hemisferio cerebral contralateral, obtenidas de médula ósea siguiendo el protocolo del Ejemplo 1 [1.1].4.2 Injections in injured brains of adult animals To perform this test, demyelinating lesions are caused in mice located in the cerebral hemisphere by stereotactic injection of lisolecithin. To do this, by means of a stereotaxic trephine, 1 μl of lysolecithin (Sigma) is slowly and slowly inoculated with the teleencephalic brain parenchyma with a Hamilton syringe. An attempt is made to locate the lateral region of the hemispheric white substance on one side of the brain. The pathological study shows that in one week there has been an important demyelination with abundant astroglial proliferation in the region of inoculation. After locating the lesion, a composition comprising a suspension of hematopoietic stem cells is injected into a physiologically acceptable vehicle, in the contralateral cerebral hemisphere, obtained from bone marrow following the protocol of Example 1 [1.1].
En animales control (animales que tenían la lesión pero que no recibieron tratamiento con células madre hematopoyéticas) se observa en la región lesionada, una semana después de localizar la lesión, una reacción astroglial acompañada de pérdida de marcadores de oligodendroglia (PLP) , así como la presencia en su periferia de células con marcadores de progenitores de oligodendrocitos (04).In control animals (animals that had the lesion but were not treated with hematopoietic stem cells), an astroglial reaction accompanied by loss of oligodendroglia (PLP) markers is observed in the injured region, one week after locating the lesion. the presence in its periphery of cells with markers of oligodendrocyte progenitors (04).
Por el contrario, en los animales lesionados y transplantados (tratados con células madre hematopoyéticas) se observan abundantes progenitores de oligodendrocitos (04+) procedentes del donante que se acumulan periféricamente e invaden la región desmielinizada.On the contrary, in injured and transplanted animals (treated with hematopoietic stem cells) abundant oligodendrocyte progenitors (04+) from the donor are observed that accumulate peripherally and invade the demyelinated region.
Los resultados obtenidos ponen de manifiesto que las células madre hematopoyéticas inyectadas en cerebros de mamíferos adultos con lesiones desmielinizantes pueden migrar atraídos por la región lesionada, invadir la lesión y generar células de la línea oligodendroglial .The results show that hematopoietic stem cells injected into the brains of adult mammals with demyelinating lesions can migrate attracted by the injured region, invade the lesion and generate oligodendroglial line cells.
4.3 Inyección intraparenteral de células madre hematopoyéticas4.3 Intraparenteral injection of hematopoietic stem cells
En animales neonatales (ratones PO) y adultos lesionadosIn neonatal animals (PO mice) and injured adults
(ratones adultos con lesiones desmielinizantes con lisolecitina) se inyecta una composición que comprende una suspensión de células madre hematopoyéticas en un vehículo fisiológicamente aceptable (2.000.000 de células en 100 microlitos de solución RPMI 1640) por vía intravenosa o intraperítoneal (células de médula ósea según el Ejemplo 1(adult mice with demyelinating lesions with lysolecithin) a composition comprising a suspension of hematopoietic stem cells is injected into a physiologically acceptable vehicle (2,000,000 cells in 100 microliths of RPMI 1640 solution) intravenously or intraperitoneally (bone marrow cells) according to Example 1
[1.1]). Al cabo de una o dos semanas tras detectar la lesión y de haber inyectado las células madre hematopoyéticas, se observa, en la región lesionada, la presencia de células del donante que muestran características neurales (astrocitos, oligodendrocitos y neuronas) . Estos resultados ponen de manifiesto que células madre hematopoyéticas inyectadas intraparenteralmente (por vía intravenosa o intraperitoneal) pueden llegar a las lesiones cerebrales, invadir el área lesionada y generar células neurales, restaurando las células perdidas . En el caso de lesiones desmielinizantes, se generan progenitores de oligodendrocitos. Según resultados previos de los propios inventores, cabe pensar, aunque no se desea estar vinculado por ninguna teoría, que esta regeneración de poblaciones perdidas se realiza mediante una transdiferenciación previa a células madre neurales. [1.1]). After one or two weeks after detecting the lesion and having injected the hematopoietic stem cells, the presence of donor cells showing neural characteristics (astrocytes, oligodendrocytes and neurons) is observed in the injured region. These results show that hematopoietic stem cells injected intraparenterally (intravenously or intraperitoneally) they can reach brain lesions, invade the injured area and generate neural cells, restoring lost cells. In the case of demyelinating lesions, oligodendrocyte progenitors are generated. According to previous results of the inventors themselves, it is possible to think, although it is not desired to be linked by any theory, that this regeneration of lost populations is carried out by prior transdifferentiation to neural stem cells.

Claims

REIVINDICACIONES
1. Un procedimiento para la producción de células madre neurales que comprende cultivar células madre hematopoyéticas bajo condiciones que promueven el crecimiento y la diferenciación de dichas células madre hematopoyéticas en células madre neurales.1. A process for the production of neural stem cells comprising culturing hematopoietic stem cells under conditions that promote the growth and differentiation of said hematopoietic stem cells into neural stem cells.
2. Procedimiento según la reivindicación 1, en el que dichas células madre hematopoyéticas se obtienen a partir de médula ósea o sangre periférica de un mamífero adulto o a partir del cordón umbilical humano.2. A method according to claim 1, wherein said hematopoietic stem cells are obtained from bone marrow or peripheral blood of an adult mammal or from the human umbilical cord.
3. Procedimiento según la reivindicación 1, en el que dichas células madre hematopoyéticas se cultivan in vi tro para obtener células madre neurales.3. A method according to claim 1, wherein said hematopoietic stem cells are cultured in vitro to obtain neural stem cells.
4. Procedimiento según la reivindicación 3, que comprende sembrar dichas células madre hematopoyéticas en un medio de cultivo, incubarlas bajo condiciones que permiten el crecimiento celular y ensayar los cultivos para detectar la presencia de células que manifiestan marcadores específicos de células madre neurales.4. A method according to claim 3, comprising planting said hematopoietic stem cells in a culture medium, incubating them under conditions that allow cell growth and testing the cultures to detect the presence of cells that manifest specific markers of neural stem cells.
5. Procedimiento según la reivindicación 1, en el que dichas células madre hematopoyéticas se cultivan in vivo para obtener células madre neurales.5. A method according to claim 1, wherein said hematopoietic stem cells are cultured in vivo to obtain neural stem cells.
6. Procedimiento según la reivindicación 5, que comprende administrar a un animal una composición fisiológicamente aceptable que comprende células madre hematopoyéticas y ensayar la presencia de células que manifiestan marcadores específicos de células madre neurales en el animal receptor. Method according to claim 5, which comprises administering to an animal a physiologically acceptable composition comprising hematopoietic stem cells and testing the presence of cells that manifest specific markers of neural stem cells in the recipient animal.
7. Procedimiento según la reivindicación 6, en el que la administración al animal de dichas células madre hematopoyéticas se realiza mediante inyección intracerebral o intraparenteral.7. A method according to claim 6, wherein the administration of said hematopoietic stem cells to the animal is carried out by intracerebral or intraparenteral injection.
8. Una composición farmacéutica que comprende células madre hematopoyéticas y un vehículo farmacéuticamente aceptable .8. A pharmaceutical composition comprising hematopoietic stem cells and a pharmaceutically acceptable carrier.
9. Empleo de células madre hematopoyéticas en la elaboración de una composición farmacéutica para el tratamiento de enfermedades neurodegenerativas y/o de situaciones patológicas en las que existen células neuronales degeneradas, tanto en procesos localizados como multifocales .9. Use of hematopoietic stem cells in the development of a pharmaceutical composition for the treatment of neurodegenerative diseases and / or pathological situations in which there are degenerated neuronal cells, both in localized and multifocal processes.
10. Empleo según la reivindicación 9, en el que dicha composición fisiológicamente aceptable se administra mediante inyección intracerebral o intraparenteral.10. Use according to claim 9, wherein said physiologically acceptable composition is administered by intracerebral or intraparenteral injection.
11. Empleo según la reivindicación 9, en el que dicha enfermedad neurodegenerativa se selecciona entre esclerosis múltiple, esclerosis lateral amiotrófica, enfermedad de Parkinson, enfermedad de Alzheimer, ataxias olivo-cerebelosas degenerativas y encefalitis espongiforme humana o Enfermedad de Creutzfeld-Jacob .11. Use according to claim 9, wherein said neurodegenerative disease is selected from multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, degenerative olive-cerebellar ataxias and human spongiform encephalitis or Creutzfeld-Jacob disease.
12. Empleo de células madre hematopoyéticas en la elaboración de una composición farmacéutica para el tratamiento de regeneración neural tras traumatismo craneoencefálico o regeneración neural postquirúrgica. 12. Use of hematopoietic stem cells in the preparation of a pharmaceutical composition for the treatment of neural regeneration after head trauma or post-surgical neural regeneration.
PCT/ES2002/000253 2001-05-28 2002-05-27 Use of haematopoietic stem cells in the generation of neural stem cells WO2002096439A1 (en)

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