WO2015181253A1 - Population de cellules progénitrices neurales - Google Patents

Population de cellules progénitrices neurales Download PDF

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WO2015181253A1
WO2015181253A1 PCT/EP2015/061750 EP2015061750W WO2015181253A1 WO 2015181253 A1 WO2015181253 A1 WO 2015181253A1 EP 2015061750 W EP2015061750 W EP 2015061750W WO 2015181253 A1 WO2015181253 A1 WO 2015181253A1
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cells
neural
substantially pure
pure population
neural progenitor
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Miodrag Stojkovic
Dunja LUKOVIC
Shomi S. BHATTACHARYA
Slaven ERCEG VUKICEVIC
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Fundación Pública Andaluza Progreso Y Salud
Universidad De Kragujevac
Instituto De Salud Carlos Iii
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Definitions

  • the present invention relates to a new method which includes the use of xeno-free differentiation medium and human extracellular matrix to successfully convert hESCs (human embryonic stem cells) and Adult hiPSC cells (human induced adult pluripotent stem cells) to regional specific and transplantable neural progenitors.
  • hESCs human embryonic stem cells
  • Adult hiPSC cells human induced adult pluripotent stem cells
  • These neural progenitor cells are not exclusively characterised by said new method of preparation but also by the fact that they naturally express a specific pattern of markers which can be used to assist with their isolation and expansion.
  • the cells of the invention display an unprecedented capacity for providing, activating and/or inducing repair of damaged neural tissue.
  • These neural progenitor cells may thus be used as therapeutic agents including, without limitation, for the regeneration of tissue, particularly for regeneration of damaged neural tissue.
  • hPSCs Human pluripotent stem cells
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • neural progenitors from pluripotent stem cells holds promise for investigation of human neurogenesis, studying the development of the central nervous system (CNS) and neural diseases and has potential for cell therapy applications to treat various neurodegenerative diseases such as Parkinson's disease (Park et al., 2005; Perrier et al., 2004; Yan et al., 2005) or spinal cord injury (Keirstead et al., 2005).
  • Parkinson's disease Park et al., 2005; Perrier et al., 2004; Yan et al., 2005
  • spinal cord injury Keirstead et al., 2005.
  • Committed progenitors derived from these cells can be transplanted into the injured nervous system where they could perform therapeutic role in a number of ways: i) they may provide trophic support to host cells, ii) facilitate axonal growth or glial function, iii) secrete neurotransmitters deficient in the host, iv) differentiate into oligodendrocytes and myelinate host axons, or v) differentiate into mature neurons and form neuronal connections across disconnected populations or replace damaged neuronal circuits.
  • hPSC differentiation towards defined neural lineage involves formation of embryonic bodies (EBs) with wide heterogeneous nature (Reubinoff et al., 2001 ; Zhang et al., 2001 ), or use undefined factors such as stromal cells (Kawasaki et al., 2000; Kawasaki et al., 2002) or animal extracellular matrix to form neuroepithelial structures called "rosettes", followed by retinoic acid (RA) exposures (Lee et al., 2007; 2008; Shin et al., 2006).
  • EBs embryonic bodies
  • stromal cells Kawasaki et al., 2000; Kawasaki et al., 2002
  • RA retinoic acid
  • the majority of these cell lines are differentiated in the presence of animal feeder cell lines or animal components, which bears risk of xenogenetic pathogen cross-transfer, limiting thus their medical applications.
  • Controlled conversion into homogeneous population of neural progenitors in animal-free conditions avoiding a formation of EB is therefore a desirable approach for basic and applied scientific research. Due to many similarities and differences between hESC and hiPSC it is important to compare their potential to differentiate toward specific cells, in particular neural cells, since this might play a critical role for many applications in both basic and applied research.
  • hESCs human embryonic stem cells
  • hiPSCs human induced adult pluripotent stem cells
  • the neural progenitors give rise in vitro and in vivo to progeny representing the three major neural lineages: oligodendrocytes, astrocytes and mature electrophysiologically functional neurons.
  • the present method is the first to be both xeno-free and adherent to differentiate hPSC toward high purity neural progenitors suitable for cell therapeutic applications.
  • a first aspect of the invention refers to a method of preparing a substantially pure
  • Obtaining undifferentiated PSCs pluripotent stem cells
  • ESC epidermal stem cells
  • ECM extracellular matrix
  • step b Replacing the extracellular matrix with a xeno-free neural differentiation medium until the formation of neural-tube-like structures called rossettes; c. Separating the neural-tube-like structures of step b) and transferring them to coated plates with a defined Matrix for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium; and d.
  • step c) Disaggregating the cells of step c) and plated them to coated plated with a defined Matrix for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium; wherein the neural progenitor cells obtained in step d) express SOX1 , PAX6, ZIC1 and do not express OCT4.
  • the method further comprises the following step e): e. Expanding the cells that are selected in step (d).
  • the method further comprises the following step f): f. Confirming that the substantially pure population of neural progenitor cells that results from step (d) or from the expansion step (e) of claim 2 expresses at least the following markers: SOX1 , PAX6, ZIC1 and does not express the following marker: OCT4.
  • the xeno-free neural differentiation medium of steps (b)-(d) is the iTS medium, wherein the iTS medium is defined as comprising: DMEM/F12, Dextran (6%), human Insulin 50pg/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (0.5M) and Ascorbic acid (50Mg/ml).
  • the coated plates with a defined matrix of step d) are human laminin/polyornithin precoated plates.
  • the cultivation step with the xeno-free neural differentiation medium of (step b) is carried-out for at least 5 days, preferably for at least 7 days.
  • the cultivating step of the neural-tube-like structures in the suitable defined matrix maintained with a xeno-free neural differentiation medium is carried-out for at least 5 days, preferably for at least 7 days.
  • the disaggregated cells in the suitable defined matrix are maintained with a xeno-free neural differentiation medium for at least 5 days, preferably for at least 7 days.
  • a second aspect of the invention refers to a substantially pure population of neural progenitor cells obtained or obtainable by the method of the first aspect of the invention.
  • a third aspect of the invention refers to a pharmaceutical composition comprising the substantially pure population of neural progenitor cells of the second aspect of the invention and a pharmaceutically acceptable carrier.
  • a fourth aspect of the invention refers to the substantially pure population of neural progenitor cells of the second aspect of the invention, or the pharmaceutically composition of the third aspect of the invention, for use in therapy.
  • a fifth aspect of the invention refers to the substantially pure population of neural progenitor cells of the second aspect of the invention, or the pharmaceutically composition of the third aspect of the invention, for use in treating a neural disease or lesion such as Parkinson disease or spinal cord injury.
  • a sixth aspect of the invention refers to the substantially pure population of neural progenitor cells or the pharmaceutically composition of the second or third aspect of the invention, wherein the substantially pure population of neural progenitor cells is allogeneic.
  • a seventh aspect of the invention refers to the substantially pure population of neural progenitor cells or the pharmaceutically composition of the second or third aspect of the invention, wherein the cells are administered intravenously or intra-arterially or intracraneally.
  • An eight aspect of the invention refers to a method of preparing a substantially pure population of mature electrophysiologically functional neurons, comprising the steps of: a. Providing the substantially pure population of neural progenitor cells of the second aspect of the invention.
  • step b Differentiating the cells of step a) into mature electrophysiologically functional neurons by using a suitable differentiation medium.
  • a ninth aspect of the invention refers to a method of preparing a substantially pure population of mature functional oligodendrocytes, comprising the steps of: a. Providing the substantially pure population of neural progenitor cells of the second aspect of the invention.
  • step b Differentiating the cells of step a) into mature functional oligodendrocytes by using a suitable differentiation medium.
  • a tenth aspect of the invention refers to a method of preparing a substantially pure population of mature functional astrocytes, comprising the steps of: a. Providing the substantially pure population of neural progenitor cells of the second aspect of the invention.
  • step b Differentiating the cells of step a) into mature functional astrocytes by using a suitable differentiation medium.
  • An eleventh aspect of the invention refers to a substantially pure population of mature functional neurons, oligodentrocytes or astrocytes obtained or obtainable by the method of any of the previous aspects of the invention.
  • a twelfth aspect of the invention refers to a pharmaceutical composition comprising the substantially pure population of mature functional neurons, oligodentrocytes or astrocytes of the eleventh aspect of the invention and a pharmaceutically acceptable carrier.
  • a thirteenth aspect of the invention refers to the substantially pure population of the eleventh aspect of the invention, or the pharmaceutically composition of the twelve aspect of the invention, for use in therapy.
  • a fourteenth aspect of the invention refers to the substantially pure population of the eleventh aspect of the invention, or the pharmaceutically composition of the twelve aspect of the invention, for use in treating a neural disease or lesion such as Parkinson disease or spinal cord injury.
  • a fifteenth aspect of the invention refers to the substantially pure population or the pharmaceutically composition of the eleventh or twelve aspect of the invention, wherein the substantially pure cell population is allogeneic.
  • a sixteenth aspect of the invention refers to the substantially pure population or the pharmaceutically composition of the eleventh or twelve aspect of the invention, wherein the cells are administered intravenously or intra-arterially.
  • a seventeenth aspect of the invention refers to the use of a xeno-free neural differentiation medium for preparing a substantially pure population of neural progenitor cells from PSCs (pluripotent stem cells), preferably from ESC (embryonic stem cells) or Adult PSCs (adult pluripotent stem cells).
  • PSCs pluripotent stem cells
  • ESC epidermal stem cells
  • Adult PSCs adult pluripotent stem cells
  • the xeno-free neural differentiation medium is the iTS medium, wherein the iTS medium is defined as comprising: DMEM/F12, Dextran (6%), human Insulin 50pg/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (0.5M) and Ascorbic acid (5C ⁇ g/ml).
  • FIGURES Figure 1 Initial differentiation of hPSC to neural progenitors. Schematic representation of the different steps in the feeder-free and animal free and chemically defined medium conditions (see Material and Methods).
  • FIG. 1 Initial differentiation of hPSC to neural progenitors, (a) Schematic representation of initial steps in the feeder-free and animal free and chemically defined medium conditions, (b) The immunocytochemistry analysis of hESC colonies treated with ITS or ECM medium at day 3 and day 7 of differentiation protocol. At day 7 all OCT4+ cells were converted to PAX6+ cells while 50% of ECM treated colony were still OCT4+.
  • FIG. 3 Immunocytochemical characterizations of hESC- (a-d) and ihPSC-(e-h) derived neuronal precursors at D21.
  • Neural progenitors were analyzed for following antibodies: SOX1/PAX6, Z01/PAX6 and SSEA4/SOX1 (a,e), BF1/PAX6, OTX2/PAX6 and SOX2/NESTIN (b,f) , TUJ1/MUSASHI, DACH, A2B5 (c,g) and PHH3, AP2/PAX6 and P75/Ki67 (d,h).
  • Neural progenitors at day 21 express neural rosettes markers (i), and genes proposed to be characteristic for FGF/EGF-expanded cells (j).
  • Neural progenitors express wide range of anterior and posterior markers (k). Magnifications: (A) 10 ⁇ , (B, C) 20 ⁇ , (D) 40 ⁇ .
  • the neural progenitors exhibit stable proliferation capacity and extensive telomerase activity, (a) Growth curves during 80 passages, (b) RT-PCR of TERT of undifferentiated and hPSC on day 21.
  • the neural progenitors maintain their neural characteristics through passages with stable expression of PAX6 (c) and SOX1 (d) maintaining low expression of endodermal (SOX17, e) and mesodermal (BRACHYURY, f).
  • Neural progenitors are functional neurons and respond to regional specific cues, (a) Functional characterization of hESC, ihPSC and immature neurons, i-iii) Current-clamp recording of action potentials evoked by current injection, iv-vi) Whole-cell voltage-clamp recording of Na+ and K+ currents. Voltage pulses from -50 mV to +70 mV.
  • derived neurons were immature motoneurons expressing ISL. Only a few cells were positive for mature motoneuron marker HB9. About 34% of generated cells were astrocytes expressing GFAP marker, (h) Neural progenitors maintained 3 weeks in NPM medium containing TIT and EGF expressed PLP marker and were positive for oligodendrocyte marker GALC. Data were averaged and represented as means ⁇ S.E.M. Magnifications: (A, B, D) 20 ⁇ , (C, F) 10 ⁇ .
  • the present invention provides a method of preparing neural progenitor cells and/or a substantially pure population of neural progenitor cells.
  • Many methods are known in the art for the preparation of neural progenitor cells. However, none of them uses a xeno-free differentiation medium and extracellular matrix to successfully convert hESC or Adult hiPSCs such as Adult hiPSC (Adult human induced pluripotent stem cells) cells to regional specific and transplantable neural progenitors.
  • the inventors have identified a number of method steps employing a xeno- free neural differentiation medium and an extracellular matrix capable of providing a substantially pure population of neural progenitor cells, and have thus developed a method of preparing said population that reduces the risk of xenogenetic pathogen cross-transfer, which comprises the steps of: (a) Obtaining undifferentiated ESCs, preferably hESCs, or Adult PSCs, preferably Adult hiPSCs, maintained on feeders with an extracellular matrix;
  • step b) Separating the neural-tube-like structures of step b) and transferring them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium; and
  • step d) Disaggregating the cells of step c) and plated them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium; wherein the neural progenitor cells obtained in step d) express SOX1 , PAX6, ZIC1 and do not express OCT4 and SSEA4.
  • a defined matrix preferably a human defined matrix
  • the cultivation step with the xeno-free neural differentiation medium of step b) is carried-out for at least 5 days, preferably for at least 7 days.
  • the cultivating step of the neural-tubelike structures in human defined matrix coated plates maintained with a xeno-free neural differentiation medium is carried-out for at least 5 days, preferably for at least 7 days.
  • the disaggregated cells plated in human defined matrix coated plates are maintained with a xeno-free neural differentiation medium for at least 5 days, preferably for at least 7 days.
  • the ESC or the Adult PSC, preferably Adult iPSCs cells, used in the method of the present invention may be isolated from a mammal, such as a rat, mouse, pig or human.
  • Adult PSCs unlike embryonic stem cells, which are defined by their origin and obtained from the inner cell mass of the blastocyst, may be isolated from any non-embryonic tissue, and will include neonates, juveniles, adolescents and adult subjects.
  • the adult PSCs of the present invention will be isolated from a non-neonate mammal, and for example from a non-neonate human, rat, mouse or pig.
  • the adult PSCs of the present invention are isolated from a human, and are therefore human adult PSCs stem cells or a substantially pure population of human Adult PSCs stem cells.
  • the ESC or Adult PSCs cells disclosed in the method of the invention may be isolated.
  • isolated indicates that the cell or cell population to which it refers is not within its natural environment.
  • the cell or cell population has been substantially separated from surrounding tissue.
  • the cell or cell population is substantially separated from surrounding tissue if the sample contains at least about 75%, in some embodiments at least about 85%, in some embodiments at least about 90%, and in some embodiments at least about 95% stem cells.
  • the sample is substantially separated from the surrounding tissue if the sample contains less than about 25%, in some embodiments less than about 15%, and in some embodiments less than about 5% of materials other than the stem cells.
  • percentage values refer to percentage by weight or by cell number.
  • the term encompasses cells which have been removed from the organism from which they originated, and exist in culture.
  • the term also encompasses cells which have been removed from the organism from which they originated, and subsequently re-inserted into an organism.
  • the organism which contains the re-inserted cells may be the same organism from which the cells were removed, or it may be a different organism, i.e. a different individual of the same species.
  • the xeno-free neural differentiation medium of steps (b)-(d) is the iTS medium.
  • the iTS medium comprises: DMEM/F12, Dextran (6%), human Insulin 50pg/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (0.5M) and Ascorbic acid (5C ⁇ g/ml).
  • the invention provides a method of preparing a substantially pure population of neural progenitor cells according to the invention, comprising the steps of: (a) Obtaining undifferentiated PSCs, preferably ESC or adult PSCs or adult iPSCs, maintained on feeders with ECM (extracellular matrix);
  • step b) Separating the neural-tube-like structures of step b) and transfer them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained in iTS medium; and
  • step d) Disaggregating the cells of step c) and plated them coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained in iTS medium; wherein the neural progenitor cells obtained in step d) express SOX1 , PAX6, ZIC1 and do not express OCT4 and SSEA4.
  • a defined matrix preferably a human defined matrix
  • the cells of step d) are disaggregated optionally by acutase and plated to human laminin/polyornithin precoated plates and maintained in ITS medium.
  • the invention provides a method of preparing a substantially pure population of neural progenitor cells according to the invention, comprising the steps of:
  • step c) Separating the neural-tube-like structures of step b) and transfer them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained in iTS medium; and (d) Disaggregating the cells of step c) optionally with acutase and plated to human laminin/polyornithin precoated plates and maintained in ITS medium; wherein the neural progenitor cells obtained in step d) express SOX1 , PAX6, ZIC1 and do not express OCT4 and SSEA4.
  • a defined matrix preferably a human defined matrix
  • any of the methods of preparing a substantially pure population of neural progenitor cells disclosed herein may comprise an additional expansion step (step e)).
  • This step is useful in increasing the size of the population of cells of the invention that are available for downstream uses such as therapeutic applications described herein.
  • the methods of the of the invention may further comprise the step, step (f), of confirming that the substantially pure population of stem cells that results from step (d) or the expansion step (e) still express at least SOX1 , PAX6 and ZIC1 and do not express OCT4 and SSEA4 by any conventional means.
  • the invention provides a method of preparing a substantially pure population of neural progenitor cells according to the invention, comprising the steps of:
  • step b) Separating the neural-tube-like structures of step b) and transferring them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium; and
  • step c) Disaggregating the cells of step c) and plated them to coated plated with a defined matrix, preferably a human defined matrix, for PSCs suitable for the maintenance of the pluripotent stem cells under feeder-independent conditions, where there are maintained with a xeno-free neural differentiation medium;
  • a defined matrix preferably a human defined matrix
  • step (e) Optionally, expanding the cells that are selected in step (d);
  • step (f) Confirming that the substantially pure population of neural progenitor cells that results from step (d) or from the expansion step (e), if any, expresses at least SOX1 ,
  • the methods of the present invention may start from any known suspension comprising a population of undifferentiated ESC or Adult PSCs.
  • the methods also include the preparation of the initial cell suspension comprising a population of undifferentiated ESC or Adult PSCs.
  • the pluripotent stem cells, especially the ESCs, used to carry out the present invention do not result from a method that implies the destruction of human embryos.
  • the substantially pure population of neural progenitor cells can be enriched by, at the end of step d), detecting and selecting those cells expressing at least SOX1 , PAX6 and ZIC1 and which do not express OCT4 and SSEA4 by any conventional means, and discarding cells that do not express these markers.
  • the population of neural progenitor cells finally obtained by any of the methods of the invention may be isolated in any suitable xeno-free medium or culture medium known in the art.
  • neural progenitor cells and neural progenitor cell population are The neural progenitor cells and neural progenitor cell population.
  • the present invention provides a new population of neural progenitor cells and/or a substantially pure population of neural progenitor cells obtained or obtainable by the methods described herein, suitable for therapeutic applications (from hereinafter the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention).
  • the invention provides a neural progenitor cell population, particularly in the form of a substantially pure population of neural progenitor cells obtained or obtainable by the methods described herein, wherein said neural progenitor cells and/or the substantially pure population of neural progenitor cells express the markers SOX1 , PAX6 and ZIC1 and do not express marker OCT4.
  • At least about 95% of the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention express all markers SOX1 , PAX6 and ZIC1 and do not express OCT4 at a detectable level. More specifically, at least about 95%, 96%, 97%, 98% 99% or 100% of the cells in the substantially pure neural progenitor cell population of the invention express all markers SOX1 , PAX6 and ZIC1 and do not express OCT4 at a detectable level.
  • the neural progenitor cells of the invention and/or cells of the substantially pure population of neural progenitor cells of the invention may also express one or more, i.e. MUSASHI, A2B5, and NESTIN.
  • substantially pure in reference to the population of cells of the invention, it is meant a population of cells, wherein the cell population essentially comprises only neural progenitor cells of the invention, i.e. the cell population is substantially pure.
  • the cell population comprises at least about 80% (in other aspects at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%) of the neural progenitor cells of the invention.
  • the term "marker” as used herein encompasses any biological molecule whose presence, concentration, activity, or phosphorylation state may be detected and used to identify the phenotype of a cell.
  • a marker In order to be considered as being expressed, a marker must be present at a detectable level. By “detectable level” is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting, immunofluorescence, ELISA or FACS analysis. “Expressed” may refer to, but is not limited to, the detectable presence of a protein, phosphorylation state of a protein or an mRNA encoding a protein.
  • a gene is considered to be expressed by a cell of the invention or a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, which corresponds to an expression level in the cell of at least about 100 copies per cell.
  • the terms "express” and "expression” have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed.
  • the comparison between the expression level of a marker in an adult stem cell of the invention, and the expression level of the same marker in another cell, such as for example a mesenchymal stem cell may be conducted by comparing the two cell types that have been isolated from the same species. Preferably this species is a mammal, and more preferably this species is human. Such comparison may conveniently be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) experiment.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the neural progenitor cells of the invention and/or the population of neural progenitor cells of the invention are characterized in that they have a distinctive expression level for certain markers.
  • the neural progenitor cells of the invention and/or the neural progenitor cell population of the invention express many specific markers at a detectable level.
  • the neural progenitor cell population of the invention is considered to express a marker if at least about 80% of the cells of the population show detectable expression of the marker. In other aspects, at least about 85%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or more of the cells of the population show detectable expression of the marker. In certain aspects, at least about 99% or 100% of the cells of the population show detectable expression of the markers.
  • the substantially pure neural progenitor cells of the invention are considered to express a marker if the expression level of the marker is greater in the cells of the invention than in a control cell.
  • greater than in this context, it is meant that the level of the marker expression in the cell population of the invention is at least 2-, 3-, 4-, 5-, 10-, 15-, 20-fold higher than the level in the control cell.
  • the neural progenitor cells of the invention and/or the substantially pure neural progenitor cell population of the invention is also characterised in that the neural progenitor cells of the invention and/or the substantially pure neural progenitor cell population of the invention do not express a particular marker or combination or markers at a detectable level. Many of these are indicative of a differentiated or partially differentiated cell. As defined herein, these markers are said be to be negative markers.
  • the substantially pure neural progenitor cell population of the invention is considered not to express a marker if at least about 80% of the neural progenitor cells or of the substantially pure adult neural progenitor cell population do not show detectable expression of the marker.
  • At least about 85%, at least about 90% or at least about 95% or at least about 97% or at least about 98% or at least about 99% or 100% of the neural progenitor cells or of the substantially pure neural progenitor cell population do not show any detectable expression of the marker.
  • lack of detectable expression may be proven through the use of an RT-PCR experiment, immunoblotting, immunofluorescence, ELISA or using FACS.
  • the markers described herein are considered not to be expressed by the neural progenitor cells of the invention, if expression cannot be reasonably detected at a level of 30 cycles of PCR, which corresponds to an expression level in the cell of less than about 100 copies per cell and/or cannot be readily detected by immunofluorescence, immunoblotting, ELISA or FACS.
  • the markers referred to in the present invention include the specific reference sequence for that marker, and any known orthologs of those markers.
  • the markers include SOX1 , PAX6 and ZIC1 and do not express OCT4 and optionally SSEA4.
  • SOX1 includes (Sex determining region Y-box 1 ) transcription factor in the Sox protein family restricted to neuroectoderm. SOX1 is involved in early central nervous system development.
  • Oct-4 includes Oct-4 and any orthologs thereof, including but not limited to Pou5f1 , POU domain class 5 transcription factor 1 , Oct-3, Oct-3/4, Oct3, Otf-3, Otf-4, Otf3- rs7, and Otf3g.
  • PAX6 (paired box 6 also named N; AN2; MGDA; WAGR; D1 1 S812E) includes at least three different protein isoforms, these being the canonical PAX6, PAX6(5a), and PAX6(APD).
  • ZIC1 (Zic family member 1 )
  • Methods of treatment, therapeutic uses and pharmaceutically acceptable compositions comprising the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention.
  • neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained or obtainable by any of the methods described herein, are suitable for use in therapy and methods of treating a neural disease, particularly cellular therapies, including the induction of tissue repair/regeneration in vivo. It is noted that the neural progenitor cells of the invention will generally be used in methods of treatment and therapeutic uses in the form of a substantially pure population of the invention.
  • the invention provides methods of treatment comprising administering the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention to a recipient subject, and also provides the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention for use in therapy.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention are useful for treating ischemic injury and neural diseases such as Parkinson, spinal cord injury, retinal distrophies, cerebellar ataxias and Alzheimer disease.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention is introduced into the body of the subject by injection or implantation.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention will be directly injected into the tissue in which they are intended to act.
  • the substantially pure population of neural progenitor cells of the invention is administered intravenously, intra-arterially or intracraneally.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be used in the regeneration of neural tissue, including in the regeneration of neurons.
  • the cells of the invention may be injected or implanted directly into the damaged neural tissue; using a needle catheter which injects the cells into the brain, intra-arterially; or intercraneally using automatic injector
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be implanted into the damaged tissue adhered to a biocompatible implant.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be adhered to the biocompatible implant in vitro, prior to implantation into the subject.
  • any one of a number of adherents may be used to adhere the cells to the implant, prior to implantation. It will be clear to a person skilled in the art, that any combination of one or more adherents may be used.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be embedded in a matrix, prior to implantation of the matrix into the subject.
  • the matrix will be implanted into the damaged tissue of the subject.
  • matrices include collagen based matrices, fibrin based matrices, laminin based matrices, fibronectin based matrices and artificial matrices. This list is
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be implanted or injected into the subject together with a matrix forming component.
  • a matrix forming component may allow the cells to form a matrix following injection or implantation, ensuring that the cells remain at the appropriate location within the subject.
  • matrix forming components include fibrin glue liquid alkyl, cyanoacrylate monomers, plasticizers, polysaccharides such as dextran, ethylene oxide-containing oligomers, block copolymers such as poloxamer and Pluronics, non-ionic surfactants such as Tween and Triton'8', and artificial matrix forming components. This list is provided by way of illustration only, and is not intended to be limiting. It will be clear to a person skilled in the art, that any combination of one or more matrix forming components may be used.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be contained within a microsphere.
  • the cells may be encapsulated within the centre of the microsphere.
  • the cells may be embedded into the matrix material of the microsphere.
  • the matrix material may include any suitable biodegradable polymer, including but not limited to alginates, Poly ethylene glycol (PLGA), and polyurethanes. This list is provided by way of example only, and is not intended to be limiting.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention will be delivered to the subject in a therapeutically effective amount. The number of cells to be delivered in vivo
  • the invention also provides a pharmaceutical composition comprising the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may comprise a cell culture medium which supports the cell viability.
  • the medium will generally be serum-free in order to avoid provoking an immune response in the recipient.
  • the carrier will generally be buffered and/or pyrogen-free.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal. This list is provided by way of illustration only, and is not intended to be limiting.
  • Solutions that are cell compositions of the invention can be prepared by incorporating cells as described herein in a pharmaceutically acceptable carrier or diluents and, as required, other ingredients enumerated above, which has been sterilized by filtration.
  • Some examples of materials and solutions which can serve as pharmaceutically- acceptable carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydro
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention may be frozen in freezing medium.
  • Any medium that preserves the viability of the cells at temperatures below about -20°C e.g. temperatures below about -40°C, or about - 80°C
  • the freezing medium may comprise 2.5% to 10% DMSO. More specifically, the freezing medium may comprise 5-7.5% DMSO.
  • Freezing medium may be based on culture medium or expansion medium described herein, further comprising foetal bovine serum or human serum or any other protein or mix of proteins able to maintain cell integrity after thawing the cells.
  • Cells of the invention can also be frozen in protein free mediums based on dextrans. After thawing, cells of invention can be washed to remove the DMSO or other freezing medium components before administration or re-suspension in administration solution. Administration solution will be any medium that preserves the viability of the cells at temperatures below about -20°C (e.g. temperatures below about -40
  • Administration solution may comprise 3-15% protein such as human serum albumin.
  • compositions of the invention may also be used in any of the methods of treatment or therapeutic uses described herein.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention can differentiate into a variety of mature neural cells such as neurons, oligodendrocytes or astrocytes. These cells can in turn be transplanted into the injured nervous system where they could perform therapeutic role in a number of ways: i) they may provide trophic support to host cells, ii) facilitate axonal growth or glial function, iii) secrete neurotransmitters deficient in the host, iv) differentiate into oligodendrocytes and myelinate host axons, or v) differentiate into mature neurons and form neuronal connections across disconnected populations or replace damaged neuronal circuits.
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention can differentiate into a variety of mature neural cells such as neurons, oligodendrocytes or astrocyte
  • neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention may be used for neuronal differentiation.
  • any suitable neuronal differentiation medium or culture medium known in the art may be used.
  • Some examples of suitable neuronal differentiation medium or culture medium are different medium commercially available
  • a particularly preferred medium is neural proliferation medium (NPM) consisted of DMEM:F-12, xeno-free B27 supplement (Invitrogen), 25 ⁇ g/ml human insulin (Sigma), 6.3 ng/ml progesterone, 10 ⁇ g/ml putrescin, 50 ng/ml sodium selenite, 50 ⁇ g/ml human holotransferin (Sigma) and supplemented with 10 um/ml all-trans-retinoic acid (NPM/RA) or supplemented with bFGF 8 ⁇ g/ml (NPM/bFGF).
  • NPM neural proliferation medium
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention can be differentiated into neuron cells by maintaining these cells in NPM supplemented with 10 um/ml all-trans-retinoic acid (NPM/RA) or bFGF 8 ⁇ g/ml (for a period of approximately 7 days after which the RA is withdrawn and the cells are maintained in NPM medium for 3 more weeks.
  • NPM medium is defined in the examples below.
  • the present invention also encompasses the neuronal cells obtained or obtainable by the differentiation of the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention. These neuronal cells may be used in any of the methods of treatment or therapeutic uses described herein. Additionally, the invention further provides a pharmaceutical composition comprising these neuronal cells. These pharmaceutical compositions may also be used in any of the methods of treatment or therapeutic uses described herein.
  • neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention may be used for oligodendrocyte differentiation.
  • any suitable oligodendrocyte differentiation medium or culture medium known in the art may be used.
  • a particularly preferred medium is NPM supplemented with 40 ng/ml triiodothyroidin (Sigma-Aldrich) and 20 ng/ml of epidermal growth factor (EGF) (Sigma-Aldrich) (MPM+Tit+EGF)
  • the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention obtained by any of the methods of the invention can be differentiated into oligodendrocytes by maintaining these cells in GRM + Tit + EGF for four weeks.
  • the present invention also encompasses the oligodendrocyte cells obtained or obtainable by the differentiation of the neural progenitor cells of the invention and/or the substantially pure population of neural progenitor cells of the invention.
  • These oligodendrocyte cells may be used in any of the methods of treatments or therapeutic uses described herein.
  • the invention further provides a pharmaceutical composition comprising the above mentioned oligodendrocyte cells. These pharmaceutical compositions may also be used in any of the methods of treatment or therapeutic uses described herein.
  • ECM was changed daily. Human pluripotent stem cells were passaged by incubation in 1 mg/ml collagenase IV (animal-free, Invitrogen) for 5-8 minutes at 37°C or mechanically dissociated and then removed to freshly prepared human foreskin fibroblast layer. Neural differentiation. Undifferentiated hESC or hiPSC were maintained on feeders with ECM medium.
  • ECM medium On day 0 ECM medium was changed to ITS medium which contains: DMEM/F12, Dextran (6%), human Insulin 50 ⁇ g/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (0.5M) and Ascorbic acid (50 ⁇ g/ml) and were maintained 7 days with daily medium changes.
  • ITS medium contains: DMEM/F12, Dextran (6%), human Insulin 50 ⁇ g/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (0.5M) and Ascorbic acid (50 ⁇ g/ml) and were maintained 7 days with daily medium changes.
  • ITS medium which contains: DMEM/F12, Dextran (6%), human Insulin 50 ⁇ g/ml, Holotransferrin 5ng/ml, Sodium Selenite (50ng/ml), Glutamax 1x, Taurine (
  • the cells were disaggregated by acutase and plated to human laminin/polyornithin precoated plates and maintained in ITS medium ( Figure 1 , 2a).
  • the rossetes were propagated and expanded in ITS medium through more 80 passages to determine proliferation and telomerase activity.
  • NPM medium consisted of DMEM:F-12, xeno-free B27 supplement (Invitrogen), 25 ⁇ g/ml human insulin (Sigma), 6.3 ng/ml progesterone, 10 ⁇ g/ml putrescin, 50 ng/ml sodium selenite, 50 ⁇ g/ml human holotransferin (Sigma).
  • the cells were maintained in NPM supplemented with 10 ⁇ /ml all-trans-retinoic acid during the following 7 days after which the RA was withdrawn and the cells were maintained in NPM during 3 weeks (Figure 5b(ii)).
  • the cells were maintained 4 weeks in NPM supplemented with 40 ng/ml triiodothyroidin (Sigma-Aldrich) and 20 ng/ml of epidermal growth factor (EGF) (Sigma-Aldrich) (Tit+EGF) ( Figure 5b (iii)).
  • EGF epidermal growth factor
  • Patch-clamp recordings For electrophysiological recordings the cells were maintained for 5 additional days in NPM. Whole-cell recordings were obtained with patch electrodes (1 .5-2.5 ⁇ ) pulled from capillary glass tubes (1 .5-1 .6 mm OD; Kimax; Kimble Products), fire-polished on a microforge MF-830 (Narishige). We used an EPC-10 patch-clamp amplifier (HEKA GmbH, Germany) and standard voltage- and current-clamp protocols designed with Patch-Master software (HEKA). In voltage-clamp configuration, squared pulse depolarization voltage steps were applied from -50 mV to +70 mV from the holding potential of -70 mV.
  • Action potentials were recorded in current-clamp configuration by applying current pulses of variable amplitude (20-100 pA). Unless otherwise specified, holding potential was -70 mV. Data were filtered at 10 kHz, digitized at a sampling interval of 20 is and stored on a Macintosh computer. Off-line data analysis was performed using lgor6. All experiments were conducted at room temperature (23-26°C). The bath solution consisted of (in mM): NaCI 140, KCI 5, CaCI 2 2, MgCI 2 2, HEPES 10, Glucose 15 (pH 7.4; osmolality 300-310 mOsm). We used two different internal (pipette) solutions.
  • Tetrodotoxin (Tocris Laboratories) was used to block TTX-sensitive voltage- dependent Na+ channels. Replacement of K+ by Cs+ in the internal solution, or external application of 4-aminopyridine (4-AP) and Tetraethylammonium chloride (TEA-CI) were applied to block outward K+ currents. Receptor antagonists including bicuculline (2 ⁇ ) (Tocris Laboratories) and 6-cyano-7-nitroquinoxaline (CNQX) (100 ⁇ ) were examined on currents mediated by neurotransmitters.
  • TTX Tetrodotoxin
  • Example 2 Results To initiate the controlled neural differentiation the ECM medium was replaced by ITS medium ( Figure 1 , 2a). At D3, the first sign of neural differentiation emerged with the typical neuroepithelial structures or rosettes in the centre of colonies ( Figure 3b), and at D5-D7 the cells organized into neural tube-like rosettes with lumens ( Figure 2b). After 7 days the cells clusters were transferred human matrix (CellStart) and maintained in ITS medium for following 7 days. For final neural differentiation the clusters were dissociated and plated to human laminin/polyornithine matrix and maintained in ITS medium for additional 7 days ( Figure 2a).
  • ITS medium There are several crucial components in ITS medium which contribute to orchestrated and highly efficient conversion of hPSC to neural progenitors. It has been shown that insulin has differentiating capacity into the neuroectodermal lineage and this effect is dependent on PI3K/AKT signalling. While sodium selenite and human holotranferrin have mainly antioxidant role in the medium Taurine could have important role in neural differentiation due its neuroprotective task.
  • the hESC- and ihPSC neural progenitors express PLZF, DACH1 , MMNR1 , PLAGL1 , NR2F1 , DMTR3, LMO3, FAM70, EVI, ZNF312, LIX1 and RSPO3 ( Figure 3c, 3g and 3i). Only a few cells were expressed protein P75 and p75 (neural crest marker) ( Figure 3d and 3h).
  • neural rosette cells exhibit neural stem cells properties similar to those previously described as NSC FGF2/EGF .
  • markers such as PMP2, HOP, S100p, SPARCL and AQP4 were strongly expressed at day 21 in all neural progenitors (Figure 3j).
  • To determine the positional identity and specification of the mature neuronal population we analyzed the expression of region-specific transcription factors at day 21 . Strong expression of BF1 and OTX2 (anterior neural markers) reveals that culture conditions promote immediate rostral neuralization of primitive ectodermal cells ( Figure 3b and 3f). The analysis revealed very heterogeneous transcription factor profile.
  • telencephalic markers FOG1 , EMX1 , EMX2 and OTX2
  • anterior hindbrain markers GBX2, HOXA1 , HOXA2 and HOXB6
  • dorsal hindbrain markers PAX7, IRX3 and PAX6
  • ventral hindbrain markers such as Nkx6.1 and NKX2.2
  • neural progenitors derived at day 21 respond to anterior- posterior and dorso-ventral instructive regionalization by exposing to morphogenic factors such as retinoic acid (RA) and bFGF during the 7 days of differentiation (Figure 3b) in animal free NPM.
  • morphogenic factors such as retinoic acid (RA) and bFGF during the 7 days of differentiation
  • Figure 3b morphogenic factors
  • the neural progenitors were also cultured in NPM with addition of Tit and EGF according to previous studies (Figure 3b). The cells were analysed by RT-PCR at day 49 of differentiation protocol.
  • TIT differentiation factor triiodothyroidin hormone
  • EGF epidermal growth factors
  • hESC and ihPSC are capable of generating mature, electrically active, neurons forming neuronal networks with functional chemical synapses.
  • the electrophysiological properties of hESC and ihPSC were analyzed after 21 to 28 days of differentiation in culture using the whole-cell configuration of the patch clamp technique.

Abstract

La présente invention concerne un nouveau procédé qui comprend l'utilisation d'un milieu de différenciation exempt de xéno-contaminants et d'une matrice extracellulaire humaine pour convertir avec succès des hESC (cellules souches embryonnaires humaines) et des cellules hi PSC adultes (cellules souches pluripotentes adultes induites humaines) en progéniteurs neuronaux régiospécifiques et transplantables. Ces cellules progénitrices neurales ne sont pas exclusivement caractérisées par ledit nouveau procédé de préparation, mais également par le fait qu'elles expriment naturellement un motif spécifique de marqueurs qui peut être utilisé pour faciliter leur isolement et leur expansion. Les cellules selon l'invention présentent une aptitude sans précédent à fournir, à activer et/ou à induire la réparation de tissu nerveux lésé. Ces cellules progénitrices neurales peuvent ainsi être utilisées en tant qu'agents thérapeutiques y compris, sans limitation, pour la régénération de tissu, notamment pour la régénération de tissu nerveux lésé.
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