WO2008064408A1 - Génération de populations enrichies de cellules progénitrices neuronales et de leur descendance - Google Patents

Génération de populations enrichies de cellules progénitrices neuronales et de leur descendance Download PDF

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WO2008064408A1
WO2008064408A1 PCT/AU2007/001821 AU2007001821W WO2008064408A1 WO 2008064408 A1 WO2008064408 A1 WO 2008064408A1 AU 2007001821 W AU2007001821 W AU 2007001821W WO 2008064408 A1 WO2008064408 A1 WO 2008064408A1
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cells
cell
population
neuronal progenitor
neural
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Brent Allan Reynolds
Geoffrey William Osborne
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The University Of Queensland
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection

Definitions

  • the invention relates to neuroscience, central nervous system (CNS) cell types and methods of isolating and enriching for selected neural cell types. More particularly, the invention relates to methods of identifying neural progenitor cells and their progeny, and generating populations of cells enriched for particular neural cell lineages.
  • CNS central nervous system
  • a characteristic shared by many disorders of the central nervous system is the loss or dysfunction of cells, particularly neurons.
  • the first report of stem cells being isolated from the adult brain was in 1992. Prior to this it was believed the adult brain was incapable of generating new cells. It is now generally accepted that stem cells are active in the adult brain and routinely generate new neurons in specific regions of the brain. Furthermore, stem cells and the cells they produce, also respond to several types of injury, such as stroke and epilepsy, by producing new cells in an attempt to repair the damaged area. However, these attempts at self-repair are often not fully successful.
  • a decline in neurogenesis may also be an underlying contributing factor to many disorders such as age related memory impairment, weight control and depression.
  • neural stem cells have been found in distinct locations distributed through the embryonic, juvenile and adult central nervous systems, in particular lining the ventricular system and within the hippocampus and subventricular zones of the adult forebrain. These cells are capable of replicating indefinitely and are multipotent, being capable of producing progeny of numerous lineages, including the neuronal lineage, and glial (astrocyte and oligodendrocyte) cell lineages.
  • neural progenitor cells are capable of proliferation but due to their restricted self renewal ability have a limited proliferative ability.
  • Progenitor cells are capable of producing all of the primary neural cell types, such as neurons or glial cells. Progenitor cells which are committed to the production of neurons are termed neuronal progenitor cells.
  • neural stem cells and their progeny have been suggested as a renewable source of one or more lineages of cells from the central nervous system.
  • neural stem cells In vitro, neural stem cells have the potential to produce large numbers of neural progeny when cultured under the control of extrinsic factors such as culture supplements or particular tissue environments. When transplanted into the brain, neural stem cell progeny or progenitor cells can survive, engraft generating new cells of defined lineages.
  • the use of neural stem cells and their progeny for the generation of large numbers of progenitor cells for cell replacement therapy (i.e. to replace cells lost to injury and diseases such as stroke, trauma, and Huntington' s disease) or as a cellular source of neuronal precursors and their differentiated progeny for in vitro assays is highly desirable.
  • stem or progenitor cells for such methods.
  • the identification and isolation of neural stem cells and neural progenitor cells has proven difficult in part because of the small number of these cells in the central nervous system relative to the number of fully differentiated cells, and because of the absence of specific definitive markers for these cells.
  • Early methods to enrich for stem or progenitor cells have relied upon their proliferative potential and their ability to differentiate into cells of different lineages in tissue culture. However/ currently available methods preferentially give rise to mixed populations of cells predominated by one glial phenotype (astrocytes) .
  • neural progenitor cells and their progeny have allowed the large scale propagation of neural progenitor cells and their progeny through the use of specific culture conditions and supplements. These cultures can then be stimulated to produce differentiated neural cells in a controlled and characterised manner through the manipulation of the culture conditions and is thought to mimic the generation of adult neuronal progenitors in vivo. While this technique provides improvements in the yield and purity of neuronal progenitors, it still produces a mixture of cells from different neural lineages with neurons representing a minority of the overall population. Where it is desirable to produce new neurons and neuron progenitors that are highly enriched, the numbers of contaminating differentiated astrocytes and oligodendroglia and their progenitors must be minimised. Similarly, where it is desirable to produce glial cells and glial progenitor cells exclusively, the numbers of neurons and neuron progenitors must be minimised.
  • extrinsic tools such as antibodies, to selectively label progenitor cells or their progeny.
  • These cells can then be isolated by techniques which recognise the marker, such as flow cytometry, or by affinity methods which utilize the marker to physically separate the cell from the unlabelled population, such as magnetic separation using antibody-coated magnetic beads, affinity chromatography or "panning" with antibody attached to a solid matrix.
  • affinity methods which utilize the marker to physically separate the cell from the unlabelled population, such as magnetic separation using antibody-coated magnetic beads, affinity chromatography or "panning" with antibody attached to a solid matrix.
  • cell labelling techniques is limited because of difficulties in providing unambiguous progenitor cell-specific markers. This is particularly true for the isolation, enrichment and purification of neuronal progenitor cells.
  • Such methods present some disadvantages; they are time consuming, they may compromise cell viability due to the additional manipulations which are required, and they may alter cell phenotype due to the cascade of cellular reactions which may be set off by antibody binding to the cell surface. It is therefore desirable to be able to identify and enrich populations of neural progenitor cells or their progeny, in particular neuronal progenitors or immature neurons, while avoiding or minimising the above-mentioned disadvantages. Such enriched populations of progenitor cells or their progeny may be utilised in studies to characterise these cells, or as a renewable source of central nervous system cells of a defined lineage for drug screening or discovery studies, and for implantation in therapeutic applications .
  • the inventors have now developed a method for identifying and enriching for neuronal progenitor cells and their progeny from a population of neural cells from the mammalian central nervous system.
  • the method is not dependent upon the use of antibody labelling or other extrinsic labelling techniques, but rather relies on the certain intrinsic properties of these cells.
  • the intrinsic properties which may be used to distinguish neuronal progenitor cells from other neural cells are selected from one or more of forward angle light scatter, side angle side scatter, autofluorescence and light polarization as evaluated in a flow cytometer.
  • a method of identifying a neuronal progenitor cell or it progeny which is present in a mixed population of neural cells comprising evaluating whether a cell possesses an intrinsic property which is characteristic of a neuronal progenitor cell.
  • the method also comprises the step of isolating the identified neuronal progenitor cell.
  • the population of neural cells comprises a mixed population sourced from a culture of cells derived from a neurosphere.
  • the culture is a neuroblast assay culture. The culture may be derived from cells from a mammalian central nervous system tissue.
  • the intrinsic property which is characteristic of a neuronal progenitor cell or its progeny is side angle light scatter. In another embodiment the intrinsic property which is characteristic of a neuronal progenitor cell or its progeny is a combination of side angle light scatter and forward angle light scatter. In another particular embodiment, the intrinsic property which is characteristic of a neuronal progenitor cell or its progeny is autofluorescence, optionally in combination with forward angle light scatter and/or side angle light scatter. In yet another particular embodiment the intrinsic property which is characteristic of a neuronal progenitor cell or its progeny is one or more of partially vertically light polarization and horizontal light polarization. In another particular embodiment the intrinsic property which is characteristic of a neuronal progenitor cell or its progeny is one or more of partially vertical light polarization and horizontal light polarization one or both of forward angle light scatter and side angle light scatter.
  • the characteristic is forward angle light scatter and side angle light scatter evaluated by flow cytometry.
  • a method of producing a population of cells which is enriched for neuronal progenitor cells and their progeny comprising evaluating with flow cytometry whether cells from a starting cell population which comprises a mixed population of neuronal progenitor cells express an intrinsic property which is characteristic of a neuronal progenitor cell or their progeny, and retaining the cells which express the intrinsic property.
  • the invention also relates to operation of a flow cytometer to identify neuronal progenitor cells and to isolate and produce populations of these cells.
  • the method also includes the step of further enriching the population of neuronal progenitor cells by the removal of cells which express astrocyte specific markers and/or by the removal of cells which express oligodendrocyte specific markers.
  • the population of cells which is enriched for neuronal progenitor cells or their progeny comprises at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% neuronal progenitor cells or their progeny.
  • isolated neuronal progenitor cells or their progeny produced according to the methods of the previous aspects.
  • the neuronal progenitor cells are human neuronal progenitor cells.
  • the neuronal progenitor cells are derived from embryonic stem cells.
  • the invention provides a method for the replacement of neurons in a patient suffering loss or dysfunction of cells or dysfunction of the central nervous system and/or for the treatment of central nervous system dysfunction, comprising transplanting a population of cells according to the invention into said patient.
  • the present inventors have established that a population of neuronal progenitor cells or their progeny can be identified from amongst a mixed population of cells of neural origin.
  • the identification utilizes intrinsic properties possessed by the neuronal progenitor cells and their progeny which distinguish them from astrocytes or oligodendrocytes and their precursors and from the less differentiated neural stem cells.
  • the identification method allows the neuronal progenitor cell population to be isolated from amongst a mixed population of neural cells, and allows the generation of cell populations which are greatly enriched for neuronal progenitor cells and their progeny.
  • a mixed population of neural cells arises when a renewable source of neuronal progenitor cells is employed, for example when cells derived from a neurosphere are cultured such as in the neuroblast assay.
  • the neuroblast assay allows the large scale propagation of neural progenitor cells and their progeny through the use of specific culture conditions and supplements. However, this technique produces a mixture of cells from different neural lineages including differentiated astrocytes and oligodendroglia and their progenitors.
  • progenitor cell refers to a cell produced during the differentiation of a stem cell which has some, but not all, of the characteristics of their terminally-differentiated progeny.
  • neuronal progenitor cell is meant to encompass a cell of neuronal lineage which is committed to differentiating into or producing progeny which are neurons.
  • Neuronal progenitor cells do not produce cells of astrocyte or oligodendrocyte lineage, and accordingly may be used as a source of new neurons .
  • the neuronal progenitor cells or their progeny which are identified using the methods described herein are further characterised as expressing the following neuronal markers; the polysialylated form of neural cell adhesion molecule (PSA-NCAM) , microtubule associated protein-2
  • MAP-2 MAP-2
  • GFAP Glial fibrillary acidic protein
  • neuronal progenitor cells or their progeny which are identified are homogeneous with respect to these markers, it may be that these cells are heterogeneous with respect to particular neuronal-specific markers. These cells may be committed to or have the potential to differentiate into different classes of neuronal cells, as exhibited by the expression of different classes of neurotransmitters.
  • isolated refers to a cell or cells which are not associated with one or more cells or one or more cellular components that are associated with the cell or cells in vivo or in vitro.
  • An "enriched population” means a relative increase in numbers of, for example, neuronal progenitor cells relative to one or more non-neuronal progenitor cell types in vivo or in vitro.
  • the neuronal progenitor cells described herein originate from mammalian central nervous system tissue.
  • the presence of neural stem cells has been identified in the central nervous systems of a wide range of mammalian species, such as primates, including human , rodents including rats, mice, guinea pigs, rabbits, and hamsters, and domesticated animals such as pigs, sheep, horses, and cattle.
  • the present inventors have identified and isolated populations of neuronal progenitor cells from both rodent and human central nervous tissue using the methods described herein, and it is anticipated that the techniques described herein will also be applicable to other mammals which have populations of neural stem cells in their central nervous systems.
  • neuronal progenitor cells which were identified and isolated in the examples and figures described herein originated from embryonic forebrain tissue, it is known that populations of stem cells and neural progenitor cells may also be found throughout the developing brain and in all regions of the adult brain in close proximity to the ventricles, such as the in the forebrain subventricular zone (SVZ) , and in the hippocampus, spinal cord and the retina. It is contemplated that populations of neuronal progenitor cells may be identified and isolated not only from these central nervous system regions but also from other central nervous tissues which contain neuronal progenitor cells, using the methods of the present invention. Indeed the methods described herein can be used to investigate whether particular regions of the central nervous system at particular developmental stages support populations of neuronal progenitor cells.
  • SVZ forebrain subventricular zone
  • Neuronal progenitor cells may be identified and selected directly from primary isolates of embryonic, juvenile or adult CNS tissue, from embryonic stem cells and from cell cultures, including using the methods described herein. It may be advantageous, however, to pre-enrich the mixed population of neural cells for neuronal progenitor cells and their progeny before identifying and selecting the neuronal progenitor cells and their progeny, particularly where it is desirable to maximise the number of neuronal cells which are selected. These techniques may increase both the numbers and the proportion of neural progenitor cells and their progeny which are available for identification and selection. Pre-enrichment of neuronal progenitor cells may be achieved using techniques which are known in the art.
  • Steindler et al. US patent application No. 2005/0153446 which is incorporated herein by reference, describes a method for the in vitro propagation and differentiation of neural precursors derived from neural stem cells of the adult brain.
  • This technique allows the cultivation of neural precursor cells attached to a solid substrate, rather than in suspension, which allows for the large scale and reproducible expansion of the precursor cells. Cells taken from these cultures may be used as the mixed population of neural cells.
  • the use of this technique may be particularly advantageous where it is desirable to produce mixed cell populations suitable for the identification and selection of neuronal progenitor cells by flow cytometry.
  • the methods established by the inventors do not require the labelling of cells with extrinsic labels, such as antibodies. Instead, the inventors have identified that particular characteristics which are inherently possessed by the neural progenitor cells and their progeny can be used to distinguish these cells from other cells in a mixed population of neural cells.
  • the methods established by the inventors are applicable to live, unfixed cells, and so can use be used to generate populations of neuronal progenitor cells and their progeny which may be cultured or which may be implanted into a recipient animal, such as a mammal.
  • the methods described herein can also be applied to populations of fixed cells.
  • SSC side angle light scatter
  • the value of the SSC parameter for any given cell has a complex basis, as it influenced by cell surface topology, the ability of cytoplasmic components to scatter light and other interactions.
  • the population of neuronal progenitor cells and their progeny exhibits a lower amount of side angle scatter than the other cell populations present in the mixed neuronal population, including the glial cell population.
  • an angle of 90° allows for an optimal detection of side angle light scatter, a person of skill in the art will recognise that variations in the angle of the side angle detector will not materially alter the evaluation of this characteristic.
  • FSC forward angle light scatter
  • Intrinsic fluorescence Another characteristic which is used to identify and select the neuronal progenitor cell population is autofluorescence, also referred to as "intrinsic fluorescence" .
  • the intrinsic levels of fluorescence associated with cells present in the mixed neural population were assessed in each fluorescence channel of a flow cytometer.
  • the neuronal progenitor population and their progeny were identified as emitting discrete levels of intrinsic fluorescence in the range of 600 to 620 nm when excited at a wavelength of 488 nm.
  • bandpass ranges of between 520 and 780 nm may be utilised to identify neuronal progenitor cells and their progeny when excited with a 488 nm excitation source.
  • the autofluorescence of neuronal progenitor cells and their progeny potentially may also be identified using other excitation wavelengths, such as 355, 405, 457, 514, 595, 633, 635, and 658 nm, although with less efficiency.
  • the population of neuronal progenitor cells and their progeny which are identified using the autofluorescence characteristic largely overlaps with the population identified using combined FSC and SSC.
  • Another characteristic which may be used to identify and select the neuronal progenitor cell population is vertical and/or horizontal light polarization. Signals are collected from vertical and horizontally, or partially horizontal and vertical, or horizontally and partially vertical polarized light at angles of either 0 or 90° relative to the orientation of the laser excitation source of 488 nm, through a bandpass filter of 488/10 nm. Partially horizontally polarised light is collected with the horizontally polarised filter at 45 degrees relative to the vertical plane of light. The population of neuronal progenitor cells and their progeny which are identified using the polarization characteristic largely overlaps with the population identified using combined FSC and SSC.
  • Modern flow cytometers provide the ability to simultaneously assess multiple parameters of individual cells at high speed.
  • the evaluation is on a combination of two or more characteristics of individual cells, for example as provided by the evaluation of both the SSC and FSC of an individual cell by flow cytometry.
  • the combination allows the selection of cells based on two or more criteria, all of which must be satisfied in order for the cells to be identified as a neuronal progenitor cell. It will be appreciated that the use of combinations of characteristics allows for more rigorous identification and selection of neuronal progenitor cells, which may produce enriched populations of greater enrichment, while potentially decreasing the yield of neuronal precursor cells and their progeny which are identified and isolated.
  • the "selection" of neuronal progenitor cells from a population of cells may be positive i.e. selecting cells possessing the desired characteristic, or negative, i.e. removing cells not possessing the characteristic, or combination of the two.
  • non-neuronal progenitor cells such as astrocytes, oligodendrocytes and their precursors are removed from the mixed population of neural cells and/or from the enriched population of neuronal progenitor cells.
  • the removal of these cells can be achieved by generally available techniques.
  • Such techniques include, but are not limited to, the use of specific ligands, such as antibodies or lectins, to bind to cell surface markers which are present on and specific to astrocytes, oligodendrocytes or their precursors, and which allow the removal of these cells, either by allowing the cells to be physically separated from the neuronal progenitor cell population or by interfering with the viability of these cells.
  • specific ligands such as antibodies or lectins
  • Figure 1 provides photomicrographs of example neuroblast assay cultures.
  • a and B illustrate that in early cultures single isolated immature neuronal stem cells can be identified on top of a bed of differentiated astrocytes.
  • C illustrates that after a few days in culture many of these single cells have begun to divide, forming small clusters of cells.
  • D illustrates that after 5-7 days in culture, distinct clusters of small immature blast cells begin to appear (see inset) .
  • E illustrates that these blast cells continue to divide, generating large clusters of cells, some of which begin to take on a more differentiated neuronal morphology (see inset) .
  • F illustrates immunocytochemistry of cultured cells after two weeks in culture, showing that many cells are immunoreactive for ⁇ Ill-tubulin (neurons) while other cells are immunoreactive for Glial Fibrillary Acidic Protein (astrocytes) .
  • Figures 2A and 2B provide an analysis of neuronal numbers by immunocytochemistry for a neuronal-specific antigen ( ⁇ -III Tubulin) which reveals that in day 8 neuroblast cultures established from first passage neurospheres from embryonic day 14 mouse forebrain, between 25-30% of the cells are neurons.
  • A shows a dual- labelling flow cytometry plot revealing the numbers of neuronal progenitor cells and their progeny as identified by ⁇ -III Tubulin (fluorescence intensity plotted on the Y axis) and glial cells as identified with GFAP (fluorescence intensity plotted on the X-axis) .
  • B provides a comparison of the frequency of ⁇ -III Tubulin positive cells in the neuroblast assay as assessed by manual counting and by flow cytometric analysis.
  • Figure 3 provides a flow cytometry analysis of the cells present in the neuroblast assay, following fixation and double labelling ⁇ -III Tubulin for neurons and GFAP for glial cells. Over 90% of the ⁇ -III Tubulin-labelled cells present in the mixed population of cells were selected in the Pl population of cells.
  • Figure 4 provides details of gates used to identify and select for a population of neuronal progenitor cells and their progeny (designated Pl) from a mixed cell population derived from the neuroblast assay. The characteristics which were used to identify and select for this population were a combination of SSC (Y-axis) and FCS (X-axis) in flow cytometry.
  • Figure 5 provides details of gates used to identify and select for a population of neuronal progenitor cells and their progeny from a mixed cell population derived from the neuroblast assay.
  • the characteristics which were used to identify and select for this population were a combination of partially vertical polarized light (Y-axis) and horizontally polarized light (X-axis) .
  • A provides an example of the gate used to select for neuronal progenitors and their progeny based on polarized light.
  • B illustrates an analysis of the cell population excluded by the gate illustrated in A using a combination of SSC (Y- axis) and FCS (X-axis) .
  • C illustrates an analysis of the population included and identified using the gate in (A) using a combination of SSC (Y-axis) and FCS (X-axis) .
  • Figure 6 provides details of a gate used to identify and select for a population of neuronal progenitor cells and their progeny from a mixed cell population derived from the neuroblast assay.
  • the characteristic which was used to identify and select for this population was autofluorescence at between 600 and 620 nm with an excitation source having a wavelength of 488 nm.
  • A illustrates the bimodal distribution of autofluorescent cells under these conditions, with the lower peak containing the neuronal progenitor cells and their progeny.
  • the X-axis corresponds to wavelength, and the Y- axis number of observed events.
  • B illustrates the FSC vs SSC analysis of this gated population of cells.
  • Figure 7 provides an analysis of the cells which are present in the Pl population, when separated from the non- Pi population by cell sorting and subsequently fixed and double labelled with ⁇ -III Tubulin for neurons and GFAP for glial cells. This figure illustrates that 70% of the Pl population were ⁇ -III Tubulin positive neuronal progenitor cells and their progeny. A proportion of these ⁇ -III Tubulin positive cells expressed processes, suggesting that they were differentiated neurons beginning to express neurites .
  • Figure 8 illustrates the further enrichment of the neuronal progenitor cells and their progeny by negative selection of glial cells.
  • a and B illustrate that the Pl population of cells post sorting can be further enriched by negative selection in the flow cytometer with antibodies to non-neuronal cell types, such as (A) an anti-04 antibody which binds to immature oligodendrocytes, or (B) the antibody A2B4, which binds to immature astrocytes and oligodendrocytes.
  • C illustrates that the use of a combination of these two negative selection antibodies allowed the production of an enriched population of neuronal progenitor cells of up to 90% purity, with confirmation that the Pl cells were of neuronal lineage using an antibody to the polysialated form of neural cell adhesion molecule.
  • Figure 9 illustrates that the transplantation of sorted neuronal progenitor cells and their progeny into the striatum of adult mice reveals that nearly all of the donor cells differentiate into neurons.
  • A shows cells which are immunoreactive for doublecortin, a marker of immature neurons.
  • B shows the transplanted cells which constitutively express Green Fluorescent Protein (driven by Tau promoter) .
  • C is a merged image which demonstrates that virtually all of the transplanted cells were neurons.
  • the inset in (B) shows high power GFP expressing donor cell with a neuronal morphology.
  • FIG 10 illustrates that the transplantation of sorted neuronal progenitor cells and their progeny into the striatum of adult mice reveal that none of the donor cells differentiate into astrocytes.
  • the transplanted GFP expressing cells have a neuronal morphology (inset) .
  • GFAP immunocytochemisty identifies astrocytes in the implant region.
  • Merged photo shows that none of the donor cells are immunoreactive for GFAP indicating that they did not differentiate into astrocytes.
  • Figure 11 illustrates sorted cells in the Pl population derived from human neural stem cells. Cell have been labelled with antibodies that recognize the neural antigen ⁇ -III Tubulin and the astrocyte antigen GFAP. The figure demonstrates that the vast majority of sorted cells are young immature neurons.
  • Figure 12 illustrates the effects of various extrinsic signalling agents and molecules on the survival of neuronal progenitors sorted from Pl population.
  • 2% fetal bovine serum (FBS) 2% fetal bovine serum (FBS)
  • 10% FBS and BMP4 (20ng/ml) caused a significant increase in the number of neurons.
  • Noggin 100 ng/ml produced a significant reduction in the numbers of surviving neurons .
  • Figure 13 provides details of gates used to identify and select for a population of cells of astrocyte lineage (designated P2) from a mixed cell population derived from the neuroblast assay.
  • the characteristics which were used to identify and select for this population were a combination of SSC (Y-axis) and FCS (X-axis) in flow cytometry.
  • SSC Y-axis
  • FCS X-axis
  • Approximately 70% of the P2 population were shown to be immunoreactive for the astrocyte specific marker GFAP. This technique therefore also allows the substantial enrichment of cells of astrocyte lineage from the mixed population of neural cells.
  • Figure 14 illustrates the stem cell frequency of the overall population, P2 population and Pl population, demonstrating the utility of using a negative selection criteria to remove undifferentiated stem cells from a population of neural precursors .
  • Neurosphere cultures were prepared from embryonic through to adult rodent or human tissue. Tissue was dissected and treated as follows: A. Embryonic Cultures:
  • Tissues were re-suspended in 2 ml of complete neural stem cell medium (NeuroCult basal medium plus NeuroCult proliferation supplement together with 20ng/ml of epidermal growth factor, all from StemCell Technologies, Vancouver, Canada)
  • Tissue was minced into spall pieces with a scalpel blade until only very small pieces remained.
  • the tissue was incubated at 37 0 C for approximately 7 minutes, depending on the amount of tissue and on its consistency. 4. At the end of the enzymatic incubation, an equal volume of trypsin inhibitor was added to halt enzymic digestion.
  • tissue suspension was then pelleted by centrifugation at 110 g for 5 minutes and virtually all of the supernatant overlaying the pellet discarded.
  • the pellet was resuspended in 2 ml of Phosphate Buffered Saline and the tissue dissociated by triturating 10-20 times using a sterile, fire- polished, cotton-plugged glass Pasteur pipette. Let the suspension settle down for 3-4 minutes.
  • the pellet was resuspended in complete culture medium so as to bring the total volume of the resulting cell suspension to 0.5 ml, and viable cells counted using trypan blue.
  • Cells were seeded at a density of 3500 viable cells/cm 2 in DMEM/F12 media with N2 supplement, in untreated 6-well tissue culture dishes (3 ml volume) or 25 cm 2 -tissue culture flasks (5 ml volume) .
  • Cells were incubated at 37 0 C, 5% CO 2 in a humidified incubator. Cells proliferated to form spherical clusters that eventually lifted off from the culture substrate as they grew larger. These primary spheres were ready for sub-culturing 7-10 days after plating, depending on the growth factors used.
  • Neurospheres were collected and dissociated into a single cells suspension and plated at a density of 50,000 to 300,000 cells/ml in uncoated T75 plastic tissue culture dishes in DMEM/F12 media with N2 supplement, together with 5% FCS and 20ng/ml of EGF and IOng/ml of bFGF.
  • DMEM/F12 media with N2 supplement, together with 5% FCS and 20ng/ml of EGF and IOng/ml of bFGF.
  • FCS DMEM/F12 N2 supplemented media containing 2% FCS.
  • Four to 6 days after switching significant numbers of immature neurons could be identified laying on top of a bed of flat protoplasmic astrocytes (see Figures IE and IF) .
  • Example 1 Single cell suspensions from embryonic or adult tissues are prepared as detail in Example 1. Cell density is determined and the cells are placed on ice prior to flow cytometer analysis or sorting.
  • Example 4 The identification and selection of neuronal progenitor cells and their progeny using flow cytometry
  • a cell sorting flow cytometer is configured such that cells carried in a 9Ou liquid stream are interrogated by a focussed laser beam with an excitation power of 200 milliwatts.
  • Light scattered in the identical direction to the laser light source passes a 1.5mm obscuration bar, a 10% Neutral density filter and is collected through a 488/lOnm bandpass filter by a photodiode detector.
  • the voltage of the detector is set to 330 (on a 1 to 10 volt scale).
  • light detected at 90 degrees relative to the laser excitation source . (side scatter) is measured through a 488/lOnm bandpass filter by a photomultiplier tube with a voltage of 330 volts on a 100 to 1000 volt scale.
  • a population of predominantly ⁇ -III tubulin immunoreactive cells present in the neural cell population produced in the neuroblast assay was found to be enriched for by selecting a population of unlabelled cells which exhibited a low SSC.
  • This population could be further enriched for cells of neuronal lineage by labelling the sorted population with either an astrocyte cell surface specific antibody A2B5, an oligodendrocyte cell surface specific antibody 04, or with both, and then sorting these labelled cells out of the Pl population.
  • the further enriched population comprised greater than 90% cells of neuronal lineage, as identified by immunoreactivity to the polysialated form of neural cell adhesion molecule.
  • the sorted Pl cell population was dramatically depleted in tissue stem cells, as identified using the Neural Colony Forming Cell Assay (Cat. # 05740, StemCell Technologies, Vancouver, Canada and methods set out in US patent application 20050112546 (Reynolds and Louis) , the contents of which are incorporated by cross reference.
  • the incidence of stem cells in the unsorted population was approximately 0.035%, and this was unchanged in the population of cells rejected by the sorting process.
  • the sorted Pl contained less than 0.001% stem cells. This suggests that this population may provide advantages for applications in which is it desirable to avoid or minimise the number of stem cells. Where cells are to be transplanted or implanted, for example, it is desirable to avoid the introduction of tissue stem cells which have the potential to generate neoplastic cells.
  • Pl cells were derived from embryonic day 14 murine forebrain which had undergone two passages in the neurosphere culture system and 8 days in neuroblast culture. These sorted cells were plated into ploy-L- ornithine coated 96 well plates in NeuroCult Basal Medium and NeuroCult NSC Differentiation Supplements (StemCell Technologies, Vancouver, Canada, Cat #s 05700 & 05703, respectively) and exposed to one of 1% FBS, 10% FBS, Growth Hormone (20ng/ml) , the BMP4 antagonist Noggin (200ng/ml) , BDNF (lOOng/ml) , BMP4 (20ng/ml) and CNTF (20ng/ml) . Relative to control, 2% FBS, 10% FBS and BMP4 gave a significant (p ⁇ 0.05) increase in the number of surviving process bearing ⁇ -III tubulin immunoreactive neurons, while Noggin produced a significant decrease.
  • tissue was collected from transgenic animals which constitutively express Green Fluorescent Protein (GFP) under the control of the TAU promoter. All cells express GFP, allowing for the easy identification of transplanted cells. Sorted populations were collected in sterile FACS tubes containing sterile PBS with 1% BSA and cell count with a hemacytometer was performed. Cells were spun at 800 rpm for 5 mins . , supernatant removed and cells resuspended in 100 - 200 ⁇ l so as to give a density of 10,000 to 50,000 cells/ ⁇ l. The donor cells were transplanted into selected sites in the brain of normal, healthy neonate or adult CDl or C57BL/6 mice or adult Wistar or Sprague-Dawley rats.
  • GFP Green Fluorescent Protein
  • the host animals were anaesthetized with sodium pentobarbital (65mg/Kg) and placed into a stereotaxic apparatus. A skin incision was made to expose the surface of the skull or vertebrae. Injection sites were located using stereotaxic coordinates to locate the desired site. Burr holes were drilled in the skull and vertebrae at the coordinate sites.
  • a 5 ⁇ l syringe was housed on a syringe pump and attached to a stainless steel cannula (30-31 gauge) via a short length of polyethylene tubing. A small air bubble and then 4-5 ⁇ l of the desired cell suspension was drawn into the cannula. The cannula was lowered to the desired location and 1-3 ⁇ l of the cell suspension was injected at a speed of 0.1-0.5 ⁇ l/min.

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Abstract

La présente invention concerne un procédé d'identification d'une cellule progénitrice neuronale ou de sa descendance présente dans une population mixte de cellules neuronales, lequel procédé consiste à évaluer si une cellule possède une propriété intrinsèque qui est caractéristique d'une cellule progénitrice neuronale.
PCT/AU2007/001821 2006-11-27 2007-11-27 Génération de populations enrichies de cellules progénitrices neuronales et de leur descendance WO2008064408A1 (fr)

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US9354170B2 (en) 2011-02-15 2016-05-31 University Of Calcutta NIR fluorescence of heavy water

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WO2000047762A2 (fr) * 1999-02-12 2000-08-17 Stemcells, Inc. Cellule souche enrichie de systeme nerveux central et populations de geniteurs, et methodes d'identification, d'isolation et d'enrichissement de telles populations

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WO2000047762A2 (fr) * 1999-02-12 2000-08-17 Stemcells, Inc. Cellule souche enrichie de systeme nerveux central et populations de geniteurs, et methodes d'identification, d'isolation et d'enrichissement de telles populations

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Publication number Priority date Publication date Assignee Title
US9354170B2 (en) 2011-02-15 2016-05-31 University Of Calcutta NIR fluorescence of heavy water

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