WO1991009936A1 - Produit cellulaire progeniteur neuronal proliferee et procede - Google Patents

Produit cellulaire progeniteur neuronal proliferee et procede Download PDF

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Publication number
WO1991009936A1
WO1991009936A1 PCT/US1990/007630 US9007630W WO9109936A1 WO 1991009936 A1 WO1991009936 A1 WO 1991009936A1 US 9007630 W US9007630 W US 9007630W WO 9109936 A1 WO9109936 A1 WO 9109936A1
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cells
culture
medium
progenitor cells
neuron progenitor
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PCT/US1990/007630
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English (en)
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Barbara D. Boss
Dennis H. Spector
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Hana Biologics, Inc.
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Publication of WO1991009936A1 publication Critical patent/WO1991009936A1/fr

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    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin

Definitions

  • This invention relates to preparation of proliferated neuron progenitor cells and their implantation.
  • this invention relates to processes for producing proliferated neuron progenitor cells which can be induced in vivo or in vitro to develop into functional neurons which produce dopamine.
  • the nervous system contains two classes of cells: the nerve cells (or neurons) and neuroglia cells (or glia). These cells are distinguished by morphological, biochemical and functional differences. Morphologically, neurons have a cell body and projecting extensions or neurites (processes) of varying length. In vivo, neuritic extensions are further divided into axons (which transfer signals away from the neuron) and dendrites (which transfer signals to the neuron). Among many other biochemical and biophysical processes, neurons synthesize specific chemicals involved in signaling of information. In the central nervous system (CNS) glia are nine times more prevalent than nerve cells. Glia are thought to serve as neural supportive elements by providing nutrients, growth or survival factors and extracellular matrices. These cells are morphologically distinct from nerve cells and do not synthesize neurotransmitters.
  • CNS central nervous system
  • tissue derived from CNS areas which are no longer displaying neuronal division in vivo will only support glial survival in vitro (see, for example, Hansson et al., Brain Research 300:9-18 [1984]) and (2) tissue derived from CNS areas still undergoing neuronal division in vivo will allow both neuronal and glial survival in vitro; those neurons will not proliferate, but can differentiate to varying degrees under in vitro conditions (see for example, Ahnert-Hilger et al., Neuroscience 17(1): 157-165 [1986] and Boss et al., Dev. Brain Res. , 36:199-218 [1987]).
  • This invention is based on the development of procedures for isolation and proliferation of neuron progenitor eel is and is directed to growth, storage, production and implantation of proliferated neuron progenitor cells.
  • the isolation and culture methods are designed to proliferate neuron progenitor cells in vitro to produce a culture which differentiates to produce dopamine-producing cells.
  • the progenitor cells differentiate either in vitro or in vivo, following implantation.
  • the cultures can be frozen while substantially maintaining cell viability.
  • the progenitor cell cultures send out neurites which are contained within the aggregates and therefore are not disrupted in the implantation process.
  • the neuron progenitor cells spontaneously differentiate in vitro .
  • the neuron progenitor cells can be induced to differentiate in vitro, producing a population of mature neurons which produce dopamine.
  • the products of this invention include a culture containing neuron progenitor cells.
  • the culture can be progenitor cells or aggregates of progenitor cells in a culture medium, or single or aggregated neuron progenitor cells on or dispersed in a substrate matrix. Most preferably, the cultures are suspension cultures in which the progenitor cells grow as aggregates.
  • the process of this invention for preparing neuron progenitor cells comprises obtaining ventral mesencephalon tissue from a mammalian donor at the appropriate stage of embryonic development; dissociation of the tissue to obtain single cells and small cell clusters for culture; culturing the neuron progenitor cells in an initial culture medium which selects for a novel cell culture containing neuron progenitor cells (adaptive period) and growing the cells for a period of time (growth period) in a second medium. During the growth period, the neuron progenitor cells proliferate.
  • the progenitor cells differentiate in vivo, following implantation and can be induced to differentiate in vitro by addition of a differentiation agent; e.g., cyclic AMP.
  • the present invention is based on the development of a method for isolating, culturing and proliferating neuron progenitor cells in vitro.
  • the isolation and culture methods are designed to proliferate neuron progenitor cells in vitro to produce a culture having an increased number of neuron progenitor cells.
  • Progenitor cells in the culture can differentiate to produce dopamine-producing cells in vitro, depending on the culture period and conditions, or can differentiate in vivo, following implantation.
  • the cultures can be frozen while substantially maintaining cell viability.
  • the progenitor cell cultures send out neurites which are not disrupted in the implantation process, thus maintaining a high percentage of viable cells upon implantation.
  • a subpopulation of progenitor cells which did not differentiate in vitro differentiate in vivo and function as tyrosine hydroxyl ase-containing neurons, gaining the ability to produce functional effects about three to six months following implantation, depending on the species of the implanted tissue and of the host.
  • a culture of this invention can be used for evaluating agents which inhibit or enhance neuroblast proliferation or differentiation or for evaluating the toxicity of an agent on neurons. Aggregate cultures are particularly advantages for these purposes. Methods for evaluation of pharmacological agents using cell cultures are well known and involve combining the agent with the culture and observing the effect of various concentrations of the agent on the cultured cells.
  • the process of this invention for preparing neuron progenitor cells comprises obtaining ventral mesencephalon tissue from a mammalian donor at the appropriate stage of embryonic development; dissociating of the tissue to obtain single cells and small cell clusters for culture; culturing the neuron progenitor cells in an initial culture medium which selects for a novel cell culture containing neuron progenitor cells and maintaining the culture for a period of time in a second medium during which the neuron progenitor cells proliferate.
  • the following terms are defined as follows:
  • Cell culture or "tissue culture” refers to the maintenance of cell viability and function in vitro. It may or may not involve cell proliferation.
  • Cell proliferation is a process of cell multiplication by means of cell division. When this occurs in vitro to any significant level in monolayer cultures, it usually involves one or more subcultures.
  • Subculture means the transfer of cells from one culture vessel to another.
  • the term is synonymous with the term "passage”.
  • “Graft” is a cell culture which has been implanted into a host animal.
  • tissue is used to prepare neuron progenitor cell cultures.
  • the tissue can be from any mammalian source, conveniently from a large mammal.
  • the tissue is from a goat, cow, sheep or other commercially raised animal. Most preferred is the use of human or porcine tissue.
  • the donor embryo is in the early stages of development, prior to neurite formation.
  • the tissue is removed from the dopaminergic system of the brain.
  • the tissue is from an area which differentiates to form an area of the brain with a relatively high concentration of TH-positive neurons.
  • the tissue is from the ventral mesencephalon.
  • the objective of the tissue preparation procedure is to disperse the tissue into single cells and small aggregates (about 500 cells per aggregate) without prolonged exposure conditions that impair cell viability.
  • a description of two tissue dissociation methods, mechanical and enzymatic, are described. Mechanical dissociation is preferred since the process is as effective as enzymatic digestion and avoids cell viability impairment caused by exposure to enzymes.
  • preparation of monolayer cultures and suspension (aggregate) cultures from the dissociated cells are preferred for implantation, since the procedure allows neuron progenitor cell differentiation within the aggregates during the culture period without disruption of neurites during the implantation process.
  • the aggregate size is controlled by the culture conditions. Preferably, culture conditions are used so that the size of the aggregates remains small enough to implant by injection through a catheter. Aggregate cultures conveniently range in size from about 100 to about 1000 microns.
  • the neuron progenitor cell culture aggregates have l oci of undifferentiated cells and loci of neurons.
  • the loci of undifferentiated cells may contain rosette-like structures in which mitotic figures are often seen.
  • the loci of neurons contain TH-positive cell bodies whose neurites appear to extend to the periphery of the aggregates. Upon histological examination, the aggregates appear to be bordered by a layer of neuropil (an acellular, neurite-rich area).
  • Monolayer cultures may be advantageous for procedures in which selection of certain populations of cells is desired.
  • the selected cells can be implanted or cultured as aggregates.
  • Monolayers which will be implanted without intermediate culture as aggregates are preferably grown on a substrate which can be removed from the culture vessel and implanted, such as amniotic membranes, rather than a substrate where enzymatic digestion is required to remove the cell culture for implantation.
  • neuron progenitor cell cultures can be successfully frozen and stored at the time of dissociation or following the completion of the selection period.
  • the cultures can be induced to differentiate in vitro at any time.
  • the neuron progenitor cell cultures of this invention are biochemically distinguishable from freshly prepared ventral mesencephalon cultures.
  • a neuron progenitor cell culture of this invention produces approximately 100-fold the amount of catechol amines.
  • the cultures produce approximately ten-fold the amount of catechol amines of the non-differentiated cultures.
  • the ratios of concentrations of the three major brain catechol amines changes following differentiation.
  • cut number 4 at 90° from cut number 1, a cross- section through the neural tube anterior to the mesencephalon, releasing the ventral mesencephalon (VM). Tease away any membranes that are still attached.
  • VM ventral mesencephalon
  • An ideal VM section should be opaque, white in color, very soft in texture, and shaped roughly like a butterfly. Any trace of clear or semi-clear tissue (i.e. meninges) should be removed from the VM section.
  • HBSS Hanks Basal Salt Solution
  • the tissue is dissociated into single cells and small aggregates of cells (less than about 500 cells per aggregate).
  • the dissociation can be by enzymatic or mechanical methods, or a combination thereof. Exemplary enzymatic and mechanical methods are described below.
  • the cells are cultured as monolayers or, preferably, in suspension cultures in which the cells form aggregates (aggregate cultures). Each cell culture type is described using a different dissociation procedure. However, any dissociation procedure can be used to prepare monolayer or aggregate cultures.
  • Aggregate cultures are seeded by placing a small number of cells in a small volume of medium, preferably 1.0 ml or less, more preferably less than 0.5 ml. Not more than about 10 s cells, preferably fewer than 10 4 cells are seeded per culture. In a most preferred embodiment, from about 2.5x10 3 to about 5x10 3 cells per well are seeded in each well of a 48 well plate. Using seeding conditions that control the initial number of cells per culture tends to control the eventual size of the aggregates. In addition, use of smaller initial seeding concentrations produced smaller aggregates and enhanced the viability of the aggregates. Specifically, the absence of necrotic cells in the centers of the aggregates correlated with lower initial plating densities and maintaining smaller aggregate size.
  • the cultures are initially grown in a first culture medium which promotes the survival of neuron progenitor cells which are capable of proliferating in a serum-free, defined medium.
  • the initial culture medium can be a basal medium supplemented with serum, hormones, growth factors and trace elements.
  • the initial culture medium can be a basal medium supplemented with fetal cord serum, preferably from the same species as the cultured cells.
  • Ham's F12 with 5% fetal cord serum is an effective initial culture medium.
  • the initial culture medium is Ham's F12 with Chang's supplement C (hereinafter Chang's) or a medium having a similar composition. Chang's is preferably present at about 5% (v/v) or more.
  • the initial culture medium additionally contains glutamine at a concentration of about 2 mM and superoxide dismutase at about 100 U/ml.
  • the cultures remain in the initial culture medium for at least 4.5 days prior to culture in growth medium.
  • the growth medium is preferably added by day 7, but more preferably between days 4.5 to 5.5.
  • step 4 with a second centrifuge tube using a 130 ⁇ mesh Nitex screen (Tetko cat. no. 3-130/43). 6. Place 10 ml room temperature HBSS in a 15 ml centrifuge tube that had been rinsed with 4% bovine serum albumin (BSA) in HBSS (hereinafter HBSS/BSA) to form a BSA-coated tube. Add 0.1 ml of DNase stock. (DNase stock is Sigma cat. no. D4527 DNase at 1 mg/ml in HBSS.)
  • HN2 Medium Switch medium to HN2 Medium 5 days later by removing about half of the medium and adding that volume of HN2 medium. Then feed about twice a week with HN2 Medium by removing approximately half of the medium and replacing that volume with fresh medium. Alternatively, the initial medium can be exchanged for HN2 medium at 5 days and half or all of the medium replaced for subsequent feedings.
  • HN2 medium is a modified version of N2 medium (Bottenstein and Sato,
  • HN2 Medium contains DMEM (low glucose) :F12 1:1 v/v and the additional ingredients shown in Table 1. The formulae for DMEM and F12 are found in Tables 2 and 3, respectively.
  • HN2 medium is prepared in polycarbonate tubes and filter sterilized using Milex yellow (i.e. "protein” non-absorbing) filters (Millipore Corp., Bedford, MA). The medium is stored not more than 2 days at 4°C prior to use.
  • Milex yellow i.e. "protein” non-absorbing filters
  • Poly-1 -ornithine coated plastic Prepare plastic the day prior to use. Dilute stock poly-1 -ornith ⁇ ne (1.0 mg/ml poly-1 - ornithine, Sigma P-2533 in sterile 0.2 M borate buffer, pH 8) 1:250 v/v in sterile borate buffer. Add an appropriate volume to surface to be coated and incubate 4 hours at 37°C. Transfer plates to 4°C for overnight storage.
  • CMF-HBSS calcium-free, magnesium-free Hanks Basal Salt Solution
  • Freezing and Thawing Neural Epithel ial Cel ls The cultures can be frozen and thawed by the following procedure. Freezing Cells
  • Cells from monolayer cultures are frozen by this procedure following the same procedure used for passaging cells. Aggregates are frozen in same medium, but need not be at any critical concentration of cells. They can be thawed back out as aggregates and recultured or transplanted.
  • progenitor cells can be induced to differentiate in vitro by adding a differentiation agent to the culture medium at an effective concentration for a time sufficient for progenitor cells in the culture to differentiate. A sufficient period of time can be determined by monitoring the cultures for a significant increase in levels of TH or dopamine.
  • Differentiation agents include sodium butyrate, butyric acid, cyclic adenosine monophosphate (cAMP) derivatives, phosphodiesterase inhibitors, adenylate cyclase activators and prostaglandins. Effective levels are determined empirically by titration.
  • a preferred differentiation agent is a cAMP derivative.
  • Preferred cAMP derivatives are 8-bromo-cyclic AMP and di-butyryl-cyclic AMP (dbc-AMP). Most preferred is dbc-AMP at a concentration in the range of from about 2 to about 5 mM (final concentration in the culture medium).
  • the differentiation agent can be added to the culture medium once the cells have been in growth medium for at least about five days. Preferably, the differentiation agent is added prior to ten days in growth medium. When the growth medium is replaced during the differentiation period, the replacement medium contains the differentiation agent. Differentiation is substantially complete following at least about seven days of continuous exposure to the differentiation agent. Seven days of use of the differentiation agent is optimal. Following completion of differentiation, the differentiation agent is preferably removed. Prolonged exposure to the agent may be toxic. Following completion of differentiation, differetniated progenitor cells in the culture cease proliferation and are preferably transplanted.
  • the time required to cure Parkinsonian symptoms is shorter than when undifferentiated progenitor cell cultures are implanted.
  • Differentiated cells do not require an initial period of time in vivo to differentiate to gain the ability to produce tyrosine hydroxyl ase.
  • the neuron progenitor cell cultures can be transplanted by well known procedures for implantation of neural tissue. A preferred procedure is described below. Following transplantation, neuron progenitor cells in the resultant grafts differentiate to produce their differentiated counterparts, neurons. A subpopulation of the neurons are TH-containing neurons which produce functional effects in the host animal.
  • Cells can be transplanted by this procedure: a. Immediately after isolation from the embryo; b. After passaging; c. After growth as an aggregate culture; d. After freezing and thawing; or e. After selection from a monolayer. 2. Resuspend cells at 1-5x10 5 cells/6 ⁇ l of HBSS. Hold cells at 4°C throughout the transplantation procedure.
  • IP injections have a quicker onset of action and produce surgical anesthesia for approximately 30-45 minutes.
  • Coordinates relate to anatomical positions within the rat brain, as taken from a stereotaxic atlas. All numbers read on the stereotaxic apparatus.
  • AP anterior-posterior
  • ML medial-lateral
  • DV Dorsal-Ventral
  • the transplant coordinates are added and/or subtracted from the zero settings. Moving to these coordinates will put the needle into the anatomical area of the brain that corresponds to the transplant coordinates taken from the atlas.
  • Brain tissue which was fixed according to the procedure in Example 1 was sectioned by the following procedure.
  • All TH-positive cell bodies are counted in every third section throughout the graft using a Zeiss microscope at 100x magnification.
  • the section thickness is measured at 1000x using an oil immersion objective. Thickness in microns is found by focusing on the top of the section, recording the micrometer reading on the focusing knob of the microscope, and then focusing through the section to the bottom and computing the difference in readings. The thickness is measured in two different areas on five representative sections.
  • the cell body length is measured at 1000x magnification using an ocular micrometer or the video image analyzer.
  • corrected number (experimental cell number) • [(average section thickness) / (average cell diameter + average section thickness)]
  • 3x10 4 cells were plated per well; and, after 6-150x proliferation, the cells were transplanted (1 well/rat).
  • the cultured tissue was injected directly into the dopamine-denervated striata of host rats.
  • eleven of 20 grafted rats have shown behavioral recovery in the amphetamine- induced rotation test. Histological analysis revealed very large grafts containing numerous dopamine neurons as identified by tyrosine hydroxylase (TH) immunohistochemistry according to the procedure described in Examples 1-4.
  • TH tyrosine hydroxylase
  • the average density of the TH-positive neurons in functioning grafts was found to be greater than 100 cells/mm 3 . Most commonly, TH-positive neurons were found to be situated at the graft periphery. Occasionally, rosette-like structures were observed within the grafts.
  • Donor tissue was dissected from the ventral mesencephalon of stage 16 fetal pigs, enzymatically dissociated, and cultured as described for aggregate cultures for 7 days. 2. From day 5 to day 7, the cells were labelled with tritiated thymidine, at 0.1 ⁇ Ci/ml (New England Nuclear, 20 Ci/mM, 1 mCi/ml, Boston, MA)
  • the rats were analyzed for function by the procedure described in Strecker et al., Exp. Brain Res. 76:315- 322 (1989) during a 16-week period post-implantation.
  • Retinoic acid x10 -7 M: 0; 0.2; 1.0; 5.0 dbc-AMP (mM): 0; 0.4; 2.0; 10.0
  • tyrosine hydroxylase (TH) DNA was evaluated for the amount of tyrosine hydroxylase (TH) DNA by standard protein immuno-slot-blot techniques.
  • the cultures without differentiation agent were used as controls. All of the cultures with added retinoic acid had from about 80 to 110% of the amount of TH as the control cultures.
  • dbc-AMP the amount of TH production was significantly increased as shown below.
  • the study demonstrates that the amount of TH DNA in the cultures was significantly increased using dbc-AMP, but was unchanged using retinoic acid.
  • the most effective concentration range for dbc-AMP as a differentiation agent is from 2.0 to 5.0 mM.
  • the cell cultures analyzed included a "control" culture.
  • the control culture was freshly isolated cells from ten porcine embryos (21-day ventral mesencephalon) prepared as described in Example 5.
  • the "standard” culture was a neuron progenitor cell culture of this invention prepared as described in Example 5 from the same embryo preparation as the control culture and cultured for two weeks. That culture included approximately one embryo- equivalent of cells.
  • the third culture was a two week progenitor cell culture prepared as the standard culture and then differentiated using 5 mM dibutyryl cyclic AMP for one additional week as described in Example 8 (for a total of three weeks in culture). That culture included approximately one-third embryo- equivalents of cells.
  • the HPLC analysis determined that the freshly prepared culture did not have detectable amounts of catecholamines. (The HPLC method can detect the presence of 10 picograms of catecholamines.)
  • the standard culture had at least about 100 picograms of catechol amines (at least 100x the catecholamine content per embryo equivalent of freshly isolated cells).
  • the differentiated culture had several fold greater catecholamine content (at least 3x) than the standard culture (at least 10x the catecholamine content per embryo equivalent of the standard culture).
  • the differentiated neuron progenitor cell culture produced different proportions of the three major brain catechol amines.
  • the ratio of the concentration of dopamine to epinephrine to norepinephrine for the standard culture and the differentiated culture are shown below.

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Abstract

L'invention est basée sur le développement de techniques d'isolation et de prolifération de cellules progénitrices neuronales et concerne la croissance, le stockage, la production et l'implantation de cellules progénitrices neuronales proliférées. Les procédés d'isolation et de culture ont pour objet de faire proliférer des cellules progénitrices neuronales de mésencéphale ventral mammifère in vitro, afin de produire un bouillon de culture effectuant une différenciation afin de produire des cellules productrices de dopamine. Les produits de cette invention comprennent un bouillon de culture contenant des cellules progénitrices neuronales, de préférence cultivées sous forme d'agrégats dans des bouillons de culture en suspension. Le procédé de l'invention permettant de préparer des cellules progénitrices neuronales consiste à prélever un tissu de mésencéphale ventral sur un donneur, au stade approprié de développement embryonnaire; à dissocier le tissu afin d'obtenir des cellules individuelles ainsi que des petites grappes de cellules destinées à une mise en culture; à mettre en culture les cellules progénitrices neuronales dans un milieu de culture initial, lequel sélectionne un nouveau bouillon de culture cellulaire contenant des cellules progénitrices neuronales, et à mettre en culture les cellules pendant une certaine durée, dans un second milieu, pendant laquelle les cellules progénitrices neuronales prolifèrent.
PCT/US1990/007630 1989-12-26 1990-12-21 Produit cellulaire progeniteur neuronal proliferee et procede WO1991009936A1 (fr)

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