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WO2018185260A1 - Nouvelle matrice dérivée d'hépatocytes humains pour la différenciation de cellules souches et la réparation de tissus - Google Patents

Nouvelle matrice dérivée d'hépatocytes humains pour la différenciation de cellules souches et la réparation de tissus Download PDF

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WO2018185260A1
WO2018185260A1 PCT/EP2018/058812 EP2018058812W WO2018185260A1 WO 2018185260 A1 WO2018185260 A1 WO 2018185260A1 EP 2018058812 W EP2018058812 W EP 2018058812W WO 2018185260 A1 WO2018185260 A1 WO 2018185260A1
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cells
ihlcs
differentiation
neural
derived
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Sabrina DESBORDES
Jakob REBER
Tanja ORSCHMANN
Theresa BAECKER
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes

Definitions

  • Novel human hepatocyte-derived matrix for stem-cell differentiation and tissue repair
  • the present invention relates to the use of immature hepatocyte-like cells (iHLCs) and/or an extracellular matrix (ECM) derived from said iHLCs as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type as well as a composition comprising said iHLCs and/or said ECM as a culture system.
  • the present invention also relates to a method for preparing an iHLC derived cell-free ECM as well as a device comprising iHLCs and/or an ECM derived from said iHLC as a culture system.
  • the present invention also comprises an in vitro method for obtaining target cells of a pre-selected cell type.
  • hPSCs Human pluripotent stem cells
  • hESCs human embryonic stem cells
  • hiPSCs human induced pluripotent stem cells
  • hiPSCs have the potential to significantly contribute to tissue engineering and regenerative medicine. This potential is attributed to their unique capacity to self-renew and to differentiate into derivatives of all three germ layers (Thomson et al. 1998, Science, 282, 1 145; Takahashi et al. 2007, Cell, 131 , 861 ; and Yu et al. 2007, Science, 318, 1917).
  • hPSCs require an optimal extracellular microenvironment consisting of an extracellular matrix (ECM) as well as growth factors and cytokines.
  • ECM extracellular matrix
  • This microenvironment mimics the conditions of normal embryonic development, during which coordinated synthesis and secretion of ECM proteins direct cell proliferation, survival, differentiation as well as tissue morphogenesis (Rozario et al. 2010, Dev Biol, 341 , 126; and Hynes 2009, Science, 326, 1216).
  • hPSCs and derivatives produce their own ECM proteins, which have been used to support in vitro cell expansion and differentiation as well as in vivo tissue regeneration (Hunt et al. 2012, Exp Cell Res, 318, 1820; and Chen et al. 2007, Stem Cells, 25, 553).
  • the decellularized matrices from hPSCs have a broader spectrum of signalling capacity owing to their embryonic origin.
  • the hPSCs-derived ECMs are free from embryonic DNA and thus have reduced risk of tumor formation, significantly improving their clinical applicability.
  • the murine soluble basement membrane extracts Matrigel (BD Biosciences and Corning) and Geltrex (Thermo Fischer Scientific) are currently used for hPSCs differentiation (Kriks et al. 201 1 , Nature, 480, 547). They even remain gold standards because they provide the best efficiency for hPSCs-differentiation in vitro.
  • the matrices provide a suitable scaffold for the survival and differentiation of grafted cells in animal models.
  • the present invention provides the use of immature hepatocyte-like cells (iHLCs) and/or an extracellular matrix (ECM) derived from said iHLCs as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • ECM extracellular matrix
  • the present invention envisages a composition comprising (a) immature hepatocyte-like cells (iHLCs) and/or (b) an iHLC-derived ECM as a culture system and (c) target precursor stem cells differentiating into target cells of said pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • ECM iHLC-derived ECM
  • the present invention contemplates a method for preparing an immature hepatocyte-like cell (iHLC) derived cell-free extracellular matrix (ECM), comprising (a) cultivating iHLCs in vitro under conditions allowing for formation of an iHLC-derived extracellular matrix
  • ECM extracellular matrix
  • iHLC immature hepatocyte-like cell
  • the present invention further encompasses a device comprising immature hepatocyte-like cells (iHLCs) and/or an extracellular matrix (ECM) derived from said iHLCs as a culture system.
  • iHLCs immature hepatocyte-like cells
  • ECM extracellular matrix
  • the present invention provides an in vitro method for obtaining target cells of a pre-selected cell type, which may comprise (a) providing immature hepatocyte-like cells (iHLCs) and/or an iHLC-derived extracellular matrix (ECM), (b) adding target precursor stem cells in a suitable cell culture medium and allowing their differentiation into the target cells of a preselected cell type, (c) obtaining the target cells of said pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • ECM extracellular matrix
  • the present invention may also comprise a target cell of a pre-selected cell-type, which may be obtained or obtainable by the in vitro method described above for use in a method for cell grafting in a patient in need thereof.
  • Fig. 1 Optimized protocol for the differentiation of hiPSC to iHLCs.
  • the cells are initially differentiated on Geltrex and at day 22 passaged onto Collagen I. This is described in Example 1.
  • Fig. 2 Differentiation of hiPSC to iHLCs and their expression of different markers.
  • iHLCs also exhibit expression of marker specific for mature hepatocytes by showing expression of Albumin, a-1 -antitrypsin, CYP3A4. Nevertheless, expression of the hepatocyte marker CYP7A1 was not detected for iHLCs. Results indicate that iHLCs are defined by the expression of immature and mature hepatocyte marker. This is described in Example 2.
  • Fig. 3 Experimental set ups of the differentiation of hiPSCs to iHLCs and their differentiation on hiPSC-derived hepatocytes into target cells of a pre-selected cell type.
  • BASF refers to BDNF, ascorbic acid, SHH, FGF8, whereas BAGCT refers to BDNF, GDNF, ascorbic acid, ⁇ 3, dibutyryl cAMP (Kriks et al. 201 1 , Nature, rosette based differentiation).
  • Fig. 4 Differentiation of hiPSC on iHLCs into neural fate.
  • TH thyroid hormone
  • TuJ1 beta3 Tubulin, mature neuronal marker, red
  • markers of terminal neuronal fate acquisition (individual chanels and merge). This is described in Example 7.
  • Fig. 5 Hepatocyte-derived matrix in comparison to Geltrex-based differentiation.
  • TH green
  • FoxA2 red
  • TH green
  • FoxA2 red
  • Fig. 6 Matrix-based system: Geltrex versus hepatocyte matrix.
  • Fig. 7 No induction into neural fate for human hepatocyte cell line HepG2.
  • mDA midbrain dopaminergic
  • HiPSCs were plated either on MS5 cells (positive control), HepG2, CMs and MPCs, and observed at days 1 , 4, 5, 7 and 13 post-differentiation.
  • Neuro-epithelial rosettes were observed in the MS5 conditions at day 1 1 but not in the other conditions, where only morphological changes were detected.
  • neuro-epithelial rosettes were picked at day 1 1 and re-plated onto P/O+LAM/FN coated-plates and showed typical rosette structures at day 13. In the other conditions, no rosettes were detected at day 13 either, demonstrating a lack of neural induction. This is described in Example 11.
  • Fig. 9 Semi-quantitative PCR and quantification of midbrain dopaminergic (mDA) neuron differentiation of iPSC on MS5 and other cell types at day 11 and day 18.
  • mDA midbrain dopaminergic
  • Neural markers Otx2 and Map2 are only present in the MS5 condition. However, the neural marker Pax6 is also observed at day 18 in the HepG2 conditions, indicating that HepG2 can support early neural induction but very inefficiently. This is described in Example 11.
  • Fig. 10 Immunocytochemistry on day 11 of midbrain dopaminergic (mDA) neuron differentiation of hiPSC on MS5, HepG2 and iPSC-derived CMs and MPCs.
  • mDA midbrain dopaminergic
  • Fig. 11 Protocol of hiPSC differentiation on Geltrex +N2B27/LDN/SB (positive control), +N2B27 (negative control) and HepG2-conditioned medium. This is described in Example 12.
  • Fig. 12 Phase contrast pictures of hiPSC differentiation on Geltrex +N2B27/LDN/SB (positive control), +N2B27 (negative control) and HepG2-conditioned medium.
  • Fig. 13 Scheme of differentiation of hiPSCs to definitive endoderm.
  • hiPSCs were differentiated to iHLCs through definitive endoderm induction, which finished at day 8. This is described in Example 13. These definitive endodermal cells were further used as supporting cells for the neural differentiation of hiPSCs in comparison to MS5 support (see Fig. 14).
  • Fig. 14 Phase contrast pictures during midbrain dopaminergic (mDA) neuron differentiation of iPSC on MS5 and definitive endodermal cells.
  • Fig. 15 Immunocytochemistry on day 11 of midbrain dopaminergic (mDA) neuron differentiation of iPSC on MS5 and definitive endoderm.
  • mDA midbrain dopaminergic
  • Fig. 16 Phase pictures during mDA neuron differentiation of hiPSC on MS5 and definitive endodermal cells.
  • Fig. 17 Semi-quantitative PCR of midbrain dopaminergic (mDA) neuron differentiation of iPSC on MS5 and definitive endoderm (DE) at day 11 and day 13.
  • mDA midbrain dopaminergic
  • DE definitive endoderm
  • Fig. 18 Quantification of semi-quantitative PCR of midbrain dopaminergic (mDA) neuron differentiation of iPSC on MS5 and definitive endoderm at day 11 and day 13 (see Figure 17). Again typical markers such as Otx2, Pax6 and Sox2, as well as the neuronal marker Map2 of mature neurons are not expressed or at very low amount in hiPSCs differentiated on definitive endoderm. This is described in Example 14.
  • Fig. 19 Decellularization protocols of iHLCs and HepG2 to be used for the neural differentiation support of hiPSCs.
  • Fig. 20 Phase contrast pictures during midbrain dopaminergic (mDA) neuron differentiation of iPSC on Geltrex, HepG2 matrix and iHLC matrix.
  • Typical neural morphology was observed at day 14, even at day 6 in hiPSCs differentiated on Geltrex, HepG2 matrix, iHLC matrix except Collagen I, where the cells detached and died. This is described in Example 16.
  • Fig. 21 Immunocytochemistry on day 11 of midbrain dopaminergic (mDA) neuron differentiation of hiPSC on Geltrex, HepG2 matrix and iHLC matrix.
  • Fig. 22 Semi-quantitative PCR during midbrain dopaminergic (mDa) neuron differentiation of iPSCs on Geltrex, HepG2 matrix and iHLC matrix at day 11 , 18 and 22.
  • En1 and Nurrl key dopaminergic markers show that differentiation of hiPSCs on iHLC matrix is the most efficient culture system for dopaminergic differentiation, looking at day 22 of differentiation. This is described in Example 16.
  • Fig. 23 Quantification of semi-quantitative PCR during midbrain dopaminergic (mDa) neuron differentiation of hiPSCs on Geltrex, HepG2 matrix and iHLC matrix at day 11 , 18 and 22 (see Figure 22).
  • Fig. 24 Decellularization protocols of iHLCs and HepG2 for neural differentiation support of hiPSCs without dual SMAD inhibition.
  • Fig. 25 Phase contrast pictures during midbrain dopaminergic (mDA) neuron differentiation of iPSC on Geltrex, HepG2 matrix and iHLC matrix without dual SMAD inhibition.
  • Fig. 26 qPCR analysis of midbrain dopaminergic (mDA) neuron differentiation in a matrix-based system on Geltrex HepG2 matrix and iHLC matrix without dual SMAD inhibition.
  • Fig. 27 Decellularization protocols of iHLCs and HepG2 for neural maturation support of hiPSCs-derived neural progenitor cells (hNPCs).
  • HepG2 cells and iHLCs were decellularized as described here, to test whether the matrices support mDA neuron maturation. This is described in Example 18.
  • Fig. 28 Immunocytochemistry of midbrain dopaminergic (mDA) neuron maturation of hNPCs on P/0+ LAM/FN, HepG2 matrix and iHLC matri
  • Fig. 29 Comparison of detected peptides of core matrisome proteins and matrisome associated proteins in iHLCs, HepG2 cells, hiPSCs and MS5 by proteomic analysis.
  • Fig. 30 Quantification of core matrisome proteins in iHLCs, HepG2 cells and hiPSCs by proteomic analysis. Marked in red low peptide number. This is described in Example 19.
  • Fig. 31 Graphic presentation of quantification of core matrisome proteins in iHLCs and HepG2 cells by proteomic analysis.
  • iHLCs Beside the selected candidate proteins with neural supporting effect, iHLCs showed a high amount of Larmbl , Larnd and Lamal in comparison to HepG2 cells. This is described in Example 19.
  • Fig. 32 Quantification of matrisome associated proteins in iHLCs, HepG2 cells and hiPSCs by proteomic analysis. Marked in red low peptide number. This is described in Example 19.
  • FIG. 33 Graphic presentation of quantification of matrisome associated proteins in iHLCs and HepG2 cells by proteomic analysis.
  • matrisome associated proteins which can support DA neural induction
  • matrisome associated proteins in iHLCs were quantified in comparison to HepG2 cells.
  • iHLCs showed a high amount of FGF2, a proliferating factor, in comparison to HepG2 cells. This is described in Example 19.
  • Fig. 34 Semi-quantitative PCR in hiPSCs, HepG2 cells and iHLCs for laminin chains.
  • Fig. 35 Confirmation of candidate proteins from the proteomic analysis by semiquantitative PCR.
  • Fig. 36 Venn diagram of significantly up-regulated core matrisome and matrisome associated proteins in iHLCs in comparison to hiPSCs, HepG2 cells and MSCs.
  • Fig. 37 List of significantly up-regulated core matrisome and matrisome associated proteins in iHLCs in comparison to hiPSCs, HepG2 cells and MSCs.
  • iHLCs showed a significantly higher expression of 18 proteins compared to the control cell types (MSC, HepG2 or hiPSC). Proteins involved in either TGF3-signalling (FBN2, IGFBP2 and 5, LTBP1 ), BMP-signalling (GDF6) or CNS development (AGRN, HAPLN 1 , LAMB1 ) could be detected.
  • FBN2, IGFBP2 and 5 LTBP1
  • BMP-signalling GDF6
  • CNS development AGN, HAPLN 1 , LAMB1
  • proteins which were only up-regulated relative to two control cell types, could be also assigned to either TGF3-signalling or CNS development.
  • SFRP1 significantly up-regulated in relative to MSC and HepG2 cells, is involved in dopamine neuron development.
  • iHLCs showed a higher expression of FBLN1 , THBS1 (TGF3-signalling) and COL3A1 , COL4A1 , TNC (CNS development) compared to hiPSC and HepG2.
  • MDK and VTN were significantly higher expressed in iHLCs in comparison to MSC and hiPSCs and are associated with CNS development as well.
  • Fig. 38 Venn diagram of significantly down-regulated core matrisome and matrisome associated proteins in iHLCs in comparison to hiPSCs, HepG2 cells and MSCs.
  • Immature HLCs were analysed by LC-MS/MS in comparison to hiPSCs, HepG2 cells and MSCs. Relations of significantly regulated proteins are illustrated by a venn-diagram. 7 proteins were significantly lower expressed in iHLCs in comparison to all control groups.
  • Fig. 39 List of significantly down-regulated core matrisome and matrisome associated proteins in iHLCs in comparison to hiPSCs, HepG2 cells and MSCs.
  • iHLCs showed a significantly lower expression of 7 proteins compared to the control cell types. Neither of these 7 proteins are involved in TGF3-, BMP- signalling or CNS development.
  • the present invention solves the limitation mentioned in the prior art by producing a human matrix to obtain therapeutic-grade cells for personalized medicine.
  • the purpose of the invention is to create a novel human matrix, which is very beneficial and efficient for standard hPSCs differentiation to therapeutically relevant cell types and for its potential use as scaffold in cell grafting. Due to its high efficiency comparable and even surpassing the mouse counterparts, in the prior art no other human matrix with this capacity may have been reported so far.
  • the human matrix of the present invention derived from cultured human hepatocytes may be used for 2D and 3D in-vitro differentiation of hiPSC into cells of clinical relevance.
  • the matrix of the present invention may have the potential to induce a neural effect by differentiating hiPSC on an extracellular matrix derived from hiPSCs-derived immature hepatocyte like cells (iHLCs).
  • Hepatocytes preferably iHLCs may be considered as a good source for hiPSC neural induction.
  • the present invention provides the use of immature hepatocyte-like cells (iHLCs) and/or an extracellular matrix (ECM) derived from said iHLCs as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • ECM extracellular matrix
  • iHLCs immature hepatocyte-like cells
  • markers of immature hepatocytes such as alpha-1 -fetoprotein (AFP), HNF4a, Nkx2.5, OATP2B1 , CYP3A7 that distinguish the cells from mature, fully developed hepatocytes or hepatocyte cell-lines (in the present invention the human cancer cell line HepG2 cells was used mainly as a model of "mature hepatocytes", yet not representing entirely a specific developmental stage), and on the other hand also expressing mature hepatocyte markers such as albumin, a-1 -antitrypsin, CYP3A4, but not expressing CYP7A1.
  • iHLCs are closer to an "immature" stage, because AFP is not lost yet. AFP is a key marker lost during terminal differentiation, therefore since iHLCs still express it, the term "immature” needs to be used herein for HLCs.
  • iHLCs Eventough being called “immature” HLCs, differentiation of target precursor stem cells into target cells of a pre-selected cell type on iHLCs and/or an ECM derived from said iHLCs is even more efficient using more "mature" iHLCs including longer iHLC differentiation such as definitive endoderm induction, followed by hepatocyte differentiation, maturation and maintenance to develop more mature iHLCs.
  • said iHLCs of the present invention are defined by expressing markers of immature and mature hepatocyte markers.
  • the specific combination of immature and mature markers in iHLCs combined with the proteomics profile / matrix proteins such as Lamal , Lama5, Lambl , Lamb2, Lamb3, Larnd , Lamc2, Perlecan, Thbsl , Nid 1 , Vcan, Ltbpl , Col5a1 , Agrn, Fbn2, Gdf6, Haplnl , Igfbp2, Igfbp5, Mdk, Vtn, Col3A1 , Col4A1 , Fbln2, Tnc, and Sfrpl of the iHLCs, which are of even more importance in terms of uniqueness as indicated elsewhere herein, may perfectly define the iHLCs of the present invention.
  • iHLCs are derived from human induced pluripotent stem cells (hiPSCs).
  • hepatocytes refer to a parenchymal liver cell of the main tissue of the liver (e.g. liver cells). Hepatocytes make up the majority of the liver's cytoplasmic mass and are involved in protein synthesis and storage, carbohydrate metabolism, cholesterol, bile salt and phospholipid synthesis and the detoxification, modification and excretion of exogenous and endogenous substances.
  • iHLCs contemplated by the present invention may be derived from human or non-human induced pluripotent stem cells (iPSCs), human or non-human adult stem cells, human parthenogenetic or non-human embryonic stem cells (ESCs), or a hepatocyte cell-line or primary hepatocytes.
  • iPSCs induced pluripotent stem cells
  • ESCs human parthenogenetic or non-human embryonic stem cells
  • hepatocyte cell-line or primary hepatocytes hepatocyte cell-line or primary hepatocytes.
  • non-human iPSCs, non-human adult stem cells or non-human ESCs being differentiated into iHLCs may also be preferred such as murine ESC-derived immature hepatocytes-like cells.
  • stem cell refers to a master cell that can reproduce indefinitely to form the specialized cells of tissues and organs.
  • a stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor cell, which then proliferates into the tissue's mature, fully formed cells.
  • stem cells can differentiate into all the specialized cells— ectoderm, endoderm and mesoderm.
  • Stem cells may be totipotent or pluripotent or multipotent and may be capable of differentiating into one or more different cell types. In mammals, two broad types of stem cells may be listed: embryonic stem cells (ESCs) and adult stem cells.
  • Embryonic stem cells refer to pluripotent stem cells and are isolated from the inner cell mass of blastocysts, which may refer to an early-stage preimplantation embryo. In general, embryonic stem cells can generate all cell types in the body, whereas adult stem cells are multipotent and can produce only a limited number of cell types.
  • plasma-parthenogenetic ESCs as used herein may refer to ESCs, which may be derived from embryos, which are not capable of developing into a human being, since the embryos are developed from an oocyte without fertilization.
  • parthenogenetic embryos are not viable, but can develop into blastocysts from which ESCs, thus parthenogenetic ESCs have been derived in mouse and human.
  • Said parthenogenetic ESCs may differentiate into hepatocytes which may be able to repopulate the liver, provide normal liver function, and/or facilitate long-term survival (Espejel et al. 2014, Stem Cells, 32(7): 1983-8).
  • totipotent refers to the ability of a cell to differentiate into any type of cell in a differentiated organism, as well as a cell of extra embryonic materials, such as placenta, etc.
  • pluripotent refers to the ability of a cell being capable of differentiating into any differentiated cell type. Pluripotent stem cells do not develop into a complete organism. A pluripotent cell does not give rise to the placenta or other tissues that are vital for foetal development. Pluripotency may distinguish embryonic stem cells from adult stem cells found in adults.
  • multipotent refers to the ability of a cell being capable of differentiating into at least two differentiated cell types.
  • hepatocyte cell-line refers to a cell line which is capable of culturing over a long period of time (i.e., years) without losing characteristic activity of a hepatocyte.
  • iHLCs which may not refer to a cell line itself, may be derived from said hepatocyte-cell line.
  • primary hepatocyte refers to a primary cell taken directly from fresh liver samples and are established for growth in vitro. Primary hepatocytes have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines thus representing a more representative model to the in vivo state.
  • iHLCs are preferably derived from human induced pluripotent stem cells (hiPSCs).
  • hiPSCs human induced pluripotent stem cells
  • iHLCs may also refer to hiPSCs-derived immature hepatocyte-like cells (iHLCs).
  • Human pluripotent stem cells also called hPSC refer to undifferentiated cells having complete differentiation versatility, i.e., the capacity to grow into any of the mammalian body's cell types, except those needed to develop a fetus. In addition, those cells have growth ability, can be self-renewing, and can remain dormant or quiescent within a tissue.
  • Human pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells (iPSCs) may be generated directly from adult cells by the iPSC technology pioneered by Shinya Yamanaka's from Kyoto, Japan in 2006.
  • induced pluripotent stem cells are preferably human induced pluripotent stem cells (hiPSCs) and may differentiate on hiPSC derived iHLCs as a co-culture system into target cells of a pre-selected cell type.
  • hiPSCs human induced pluripotent stem cells
  • the differentiation of hiPSCs into iHLCs including these key stages of liver development is described in Example 1.
  • the present invention may envisage the differentiation of hiPSC into iHLCs being determined by definitive endoderm induction.
  • Definitive endoderm induction may be more than 4 days, less than 10 days, or 4-10 days, 6-8 days, or at least 4, 5, 6, 7, or at least 8 days long.
  • definitive endoderm induction is 8 days.
  • the present invention may also provide RPMI medium comprising 1 % B27 w/o insulin supplemented with 100ng ml "1 FGF2 and 100ng ml "1 Activin A for definitive endoderm differentiation, which determines the differentiation of hiPSC into iHLCs.
  • definitive endodermal cells refer to cells from the definitive endoderm being formed during gastrulation and initially appear as a single-cell thick, epithelial sheet that covers the bottom surface of the developing embryo.
  • Definitive endodermal cells may be obtained by subjecting hiPSCs to a suitable protocol (Example 13) or definitive endoderm may be obtained by other types of pluripotent cell lines or cells showing the potential to differentiate into definitive endoderm.
  • Definitive endodermal cells may be induced in RPMI medium and/or SMEM medium, preferably in RPMI medium supplemented with Activin A and FGF2.
  • Definitive endoderm cells may be mammalian cells, preferably definitive endoderm cells are human cells.
  • Definitive endoderm cells may express or fail to significantly express certain markers, preferably one or more markers selected from SOX17, CXCR4, MLXL1 , GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1 , CMKOR1 are expressed in definitive endoderm cells.
  • markers preferably one or more markers selected from SOX17, CXCR4, MLXL1 , GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1 , CMKOR1 are expressed in definitive endoderm cells.
  • definitive endodermal cells are characterized by the expression of the following markers SOX17, FoxA2, CXCR4 and down regulation of the marker SOX7.
  • the present invention may comprise the use of iHLCs being differentiated from hiPSCs, wherein said hiPSCs may be first differentiated into definitive endodermal cells using RPMI medium supplemented with Activin A and FGF2 before differentiating into iHLCs, as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • hiPSCs after hiPSCs are differentiated to definitive endodermal cells, they may further differentiate into hepatocytes.
  • Said hepatocyte differentiation may be more than 4 days, less than 10 days, or 4-10 days, 6-8 days, or at least 4, 5, 6, 7, or at least 8 days long.
  • said hepatocyte differentiation is 8 days long.
  • the present invention may also provide DMEM/F12 (1 : 1 ) medium supplemented with 10 % KOSR, 100 ng ml "1 human hepatocyte growth factor (HGF) and/or 1 % DMSO for hepatocyte differentiation from definitive endodermal cells.
  • HGF human hepatocyte growth factor
  • Hepatocyte differentiation may be achieved by having an SOX17 expression level of more than 60%, less than 100%, or about 60%-100%, 70%-100%, 80%-100%, 90%-100% or about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100%.
  • the differentiation into said iHLCs, which is divided into maturation and maintenance may be more than 1 day, less than 10 days, or 1 -10 days, 2-8 days, 4-6 days, or at least 1 , 2, 3, 4, 5, or at least 6 days long.
  • differentiation into said iHLCs is 6 days long.
  • iHLC maturation to become fully developed iHLCs is 3 days long.
  • the present invention may also contemplate DMEM/F12 (1 : 1 ) medium supplemented with 10 "7 M Dexamethasone for said hepatic maturation to become iHLCs.
  • iHLC maintenance is preferably 3 days long.
  • the present invention may also provide William's E medium supplemented with 10% Hyclone, 'l g ml "1 human Insulin, 1 .8 % DMSO, 4.7 ⁇ g ml "1 Hydrocortisone for further iHLC maintenance (Fig. 3A and 1 ).
  • the present invention may provide the use of said iHLCs for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • target precursor stem cells refer to stem cells that are capable of differentiating into specific target cells.
  • the precursor stem cells of the present invention may be capable of differentiating into neural cells, but not restricted to these cells.
  • target cells of a pre-selected cell type refers to cells being differentiated to from a cell type being selected in advance.
  • hiPSCs which may refer to target precursor stem cells
  • hiPSCs-derived iHLCs into neural cells
  • the differentiation of hiPSCs on hiPSCs-derived iHLCs may refer to rosette-based differentiation according to Kriks et al., Nature, 201 1 .
  • Irradiated MS5 cells may further be used as a control for the differentiation of hiPSCs (Fig. 3C and Example 5).
  • Murine MS5 cells are mouse bone- marrow stromal cells and are the original co-culture system for neural induction in the literature.
  • a neuroectoderm rosette refers to neuroectodermal cells in a columnar rosette structure containing neural progenitor cells (NPCs).
  • NPCs neural progenitor cells
  • Neuroectodermal cells are cells of the ectoderm, the outermost of the three primary germ layers of an embryo, the others being the mesoderm and the endoderm, which gives rise to the central and peripheral nervous systems, including some glial cells.
  • An enhanced neuroectodermal specification may be detected by the increase of the expression of neuroectodermal markers such as Pax6 and N-Cadherin once neural differentiation is initiated, thereby leading to more NPC generations.
  • the differentiation of target precursor stem cells into target cells of a pre-selected cell type may be preferred when target precursor stem cells are preferably plated on said iHLCs in comparison to a differentiation of target precursor stem cells on MS5 cells.
  • the present invention may provide for neural induction done by rosette based differentiation of hiPSC on iHLCs KSR medium supplemented with 1 ⁇ dorsomorphine and 10 ⁇ SB431542, 100 ng ml "1 SHH C24II and 100 ng ml "1 FGF8.
  • the neural induction of hiPSC rosette based differentiation may be more than 5 days, less than 20 days, or 5-20 days, 10-20 days, 15-20 days, or at least 5, 10, 1 1 , 12, 13, 14, 15, 16, 17, or at least 18 days, preferably neural induction of hiPSC rosette based differentiation is 18 days.
  • the present invention may envisage neural differentiation done by rosette based differentiation of hiPSC on iHLCs in Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2mM L-glutamine supplemented with 100 ng ml "1 SHH C24II, 100 ng ml "1 FGF8, 20 ng ml "1 BDNF, 0.2 mM ascorbic acid (abbreviated "BASF”), followed by further differentiation in Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2mM L-glutamine supplemented with 20 ng ml "1 BDNF, 20 ng ml "1 GDNF, 0.2 mM ascorbic acid, 1 ng ml "1 ⁇ 3 and 0.5 mM dibutyryl cAMP (abbreviated "BAGC”) (Example 4).
  • BAGC mM dibutyryl cAMP
  • Differentiation into target cells of a pre-selected cell type, preferably into neural cells done by rosette based differentiation is in total more than 30 days, less than 60 days, or 30-60 days, 40-60 days, 40-55 days, 40-50 days, 45-55 days, 45-50 days, or at least 30 days, at least 40 days, at least 45 days, at least 50 days, most preferably 50 days. (Fig. 3B).
  • iHLCs functioning as feeder cells for the differentiation of target precursor stem cells into target cells of a pre-selected cell type may also induce neural fate more efficiently in comparison to HepG2 cells, hiPSCs-derived cardiomyocytes (CMs) and/or mesenchymal progenitor cells (MPCs), feeder cells not being capable of differentiating target precursor stem cells into target cells of a pre-selected cell type.
  • CMs hiPSCs-derived cardiomyocytes
  • MPCs mesenchymal progenitor cells
  • the present invention may envisage the use of an extracellular matrix (ECM) derived from said iHLCs as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • ECM extracellular matrix
  • the use of an extracellular matrix (ECM) derived from said iHLCs as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type may also be preferred in the present invention.
  • ECM extracellular matrix
  • an "ECM” as used herein may also refer to a "concentrated solution of ECM proteins", thus being highly concentrated as indicated elsewhere herein
  • Said extracellular matrix of the present invention may be characterized by the expression of Lamal , Lama5, Lambl , Lamb2, Lamb3, Larnd , Lamc2, Perlecan, Thbsl , Nidi , Vcan, Ltbpl , Col5a1 , Agrn, Fbn2, Gdf6, Haplnl , Igfbp2, Igfbp5, Mdk, Vtn, Col3A1 , Col4A1 , Fbln2, Tnc, and SfrpL
  • the ECM of the present invention may preferably be produced by iHLCs being differentiated preferably from hiPSC, thus being also called "iHLC-derived ECM".
  • iHLC matrix or "hiPSCs-derived iHLC matrix” can be used interchangeably with the term iHLC-derived ECM (a hepatocyte matrix).
  • derived or “derived from” is intended that the matrix may be obtained from iHLCs.
  • iHLCs being preferably derived from hiPSCs may be a valuable source of human ECM for in vitro differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • the present invention may also envisage an iHLCs-derived ECM, wherein said ECM may be substantially cell-free.
  • said cell-free iHLC-derived ECM may be used for the plating of undifferentiated hiPSCs and their differentiation into neural cells.
  • iHLCs may secrete extracellular molecules, which may form an ECM and which may need to be decellularized, meaning isolating the ECM from its inhabiting cells, preferably iHLCs by use of a specific decellularization buffer (Example 3).
  • This decellularized or cell-free iHLC matrix may then be used for hiPSC neural differentiation.
  • the term "decellularized” or “decellularization” refers to the process used in biomedical engineering to isolate the ECM in a cell culture from its inhabiting cells.
  • the ECM derived from said iHLCs is preferably isolated from the hiPSC derived iHLCs by a specific decellularization buffer containing 20 mM ammonium hydroxide (NH 4 OH), 0.5% Triton X-100 in PBS and steril filtered 0,22 ⁇ (Example 3).
  • the replication of the hepatocyte de-cellularization and the use of the remaining ECM derived from said hepatocytes, preferably derived from hiPSC derived iHLCs, may be considered as being important for culturing and differentiating target precursor stem cells into target cells of a pre-selected cell type.
  • hiPSCs-derived iHLCs may be a valuable source of human ECM for in vitro induction of neurons
  • the differentiation of hiPSC on hiPSCs- derived iHLC matrix into neural cells may be performed, wherein said iHLC matrix may need to be decellularized before.
  • the differentiation of hiPSCs on a cell-free iHLC matrix is illustrated in Figure 3D or described in Example 6.
  • the differentiation of hiPSCs on hiPSCs- derived iHLC matrix may refer to floor-plate based differentiation according to Kriks et al., Nature, 201 1.
  • a Geltrex matrix may further be used as a control for the differentiation of hiPSCs.
  • a Geltrex matrix refers to a soluble form of reduced growth factor (RGF) basement membrane extract (BME) purified from murine Engelbreth-Holm-Swarm tumor.
  • RGF reduced growth factor
  • BME basement membrane extract
  • LDEV lactate dehydrogenase-elevating virus
  • Floor-plate based differentiation of hiPSCs on hiPSCs-derived iHLC matrix into neural cells may be more efficient than floor-plate based differentiation of hiPSCs on mouse sarcoma-derived Geltrex matrix.
  • the differentiation of target precursor stem cells into target cells of a pre-selected cell type may be preferred when target precursor stem cells are plated on an ECM derived from said iHLCs in comparison to a differentiation of target precursor stem cells on Geltrex matrix.
  • the present invention may provide for neural induction done by floor-plate based differentiation of hiPSC on hiPSC derived iHLC matrix KSR medium supplemented with 100 nM LDN193189, 10 ⁇ SB431542, 100 ng ml "1 SHH C24II, 2 ⁇ Purmorphamine 100 ng ml "1 FGF8 and 3 ⁇ CHIR99021 (Example 6) and N2B27 1 :1 medium
  • N2 medium comprises DMEM F12, 1x N2, 5 ⁇ g ml insulin, 2 mM L-glutamine, 1 x non-essential amino acids, 10 ⁇ 2- mercaptoethanol, 50 U/ml Penicillin, 50 ⁇ g ml Streptomycin
  • B27 medium comprises Neurobasal, 1 x B27, 2 mM L-glutamine, 50 U/ml Penicillin, 50 ⁇ g/ml Streptomycin) supplemented with 100 nM LDN193189 and 10 ⁇ SB431542, with
  • Neural induction (also called floor- plate induction or FP induction) of hiPSCs done by floor-plate based differentiation with KSR medium may be more than 1 day, less than 15 days, or 1 -15 days, 5-15 days, 10-15 days, or at least 1 , 3, 5, 6, 7, 8, 9, 10, or at least 1 1 days, preferably neural induction initiated by KSR medium is 1 1 days.
  • Neural induction of hiPSCs done by floor-plate based differentiation with N2B27 1 :1 medium may be more than 1 day, less than 20 days, or 1 -20 days, 5-20 days, 10-20 days, 10-15 days, or at least 1 , 3, 5, 6, 7, 8, 9, 10,1 1 , 12, 13, or at least 14, preferably neural induction initiated by N2B27 1 :1 medium is 14 days.
  • the present invention may also envisage neural differentiation done by floor-plate based differentiation of hiPSC on iHLC matrix in Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2mM L-glutamine supplemented with 3 ⁇ CHIR99021 and 20 ng ml "1 BDNF, 20 ng ml "1 GDNF, 0.2 mM ascorbic acid, 1 ng ml "1 ⁇ 3, 0.5 mM dibutyryl cAMP, 10 ⁇ DAPT (abbreviated "BAGCT”) (Example 6 and 15).
  • BAGCT DAPT
  • Differentiation into target cells of a pre-selected cell type, preferably into neural cells done by floor-plate based differentiation is in total more than 30 days, less than 60 days, or 30-60 days, 40-60 days, 40-55 days, 40-50 days, 45-55 days, 45-50 days, or at least 30 days, at least 40 days, at least 45 days, at least 50 days, most preferably 50 days (Fig. 3D and Fig. 19).
  • the differentiation of hiPSCs on iHLC matrix in N2B27 1 :1 medium was more efficient in neural induction, preferably in induction of midbrain dopaminergic neurons (mDAs) in comparison to KSR medium.
  • mDAs midbrain dopaminergic neurons
  • the differentiation into target cells of a pre-selected cell type, preferably into neural cells, more preferably into dopaminergic neurons done by floor-plate based differentiation with N2B27 1 :1 medium is the preferred differentiation.
  • the present invention may also comprise iHLCs being characterized by the expression of a-fetoprotein (uniprot accession number P02771 ), albumin (uniprot accession number P02768), HNF4a (uniprot accession number P41235), a-1 -antitrypsin (uniprot accession number P01009), CYP3A4 (uniprot accession number P08684), CYP3A7 (uniprot accession number P24462) and do not express CYP7A1 (uniprot accession number P22680).
  • a-fetoprotein uniprot accession number P02771
  • albumin uniprot accession number P02768
  • HNF4a uniprot accession number P41235
  • a-1 -antitrypsin uniprot accession number P01009
  • CYP3A4 uniprot accession number P08684
  • CYP3A7
  • pre-hepatic cells become proliferative and begin to form the liver bud.
  • These pre-hepatic cells are also designated as hepatoblasts and a number of studies have indicated that hepatoblasts give rise to hepatocytes.
  • HNF-4a being a liver transcription factor, which controls the expression of several hepatic genes, plays an important role in the early development of the liver and is highly expressed in the precursors of hepatocytes (hepatoblasts), thus referring to a marker of immature hepatocytes (Tanaka et al. 2009, Mechanisms of Development, vol. 126, Issues 8-9, pp. 665-676).
  • markers of immature hepatocytes may also comprise besides AFP CYP3A7 to identify immature hepatocytes from mature hepatocytes characterized by the expression levels of CYP3A4, G6P, alpha-1 -antitrypsin, glutamine synthetase, CYP1 B1 and/or CK18.
  • AFP alpha-fetoprotein
  • Mature hepatocytes may also be characterized by the expression levels of apolipoprotein-F, CYP1A1 and/or CYP1A2.
  • "immature hepatocytes” as used herein may refer to a hepatocyte lineage cell that may express AFP, CYP3A7and/or HNF-4a but that does not express appreciable levels of functional albumin, CYP3A4, CYP1 B1 , G6P, alpha-1 -antitrypsin, glutamine synthetase, CK18, apolipoprotein-F, CYP1A1 and/or CYP1A2.
  • immature hepatocytes must undergo maturation to acquire the functionality of mature hepatocytes.
  • iHLCs may express albumin, a-1 -antitrypsin, CYP3A4, being a marker of mature hepatocytes, besides HNF4a, Nkx 2.5, a-fetoprotein (AFP), CYP3A7 and OATP2B1 , which may refer to markers of immature hepatocytes.
  • iHLCs may not express CYP7A1 enzyme, also being a marker of mature hepatocytes (Cai et al. 2007). Said different expression signature may distinguish iHLCs from normal immature hepatocytes. Thus, these cells may be best described as an immature hepatocyte like cell.
  • iHLCs In contrast to mature hepatocytes, iHLCs preferably express AFP, which are not expressed by mature hepatocytes any longer. In contrast to HepG2 cells, iHLCs are less mature, they are not a cell line and do not express CYP7A1 , being expressed by HepG2 cells.
  • HepG2 cells refer to an immortalized, widely used human hepatocellular carcinoma cell line that is highly differentiated and displays many of the genotypic features of mature hepatocytes. Yet, since HepG2 cells is a cancer cell line, those cells are mainly a "model of mature hepatocytes", not representing entirely a specific developmental stage.
  • the present invention may comprise iHLCs preferably being characterized by an expression level of AFP of more than 50%, less than 100%, or about 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, or at least 50%, 60%, 70%, 80%, or at least 90% or about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.
  • the present invention may comprise iHLCs preferably being characterized by an expression level of albumin of more than 50%, less than 100%, or about 50%-100%, 60%-100%, 70%- 100%, 80%-100%, 90%-100%, or at least 50%, 60%, 70%, 80%, or at least 90%, or about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.
  • the present invention may comprise iHLCs preferably being characterized by an expression level of a-1 -antitrypsin of more than 50%, less than 100%, or about 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, or at least 50%, 60%, 70%, 80%, or at least 90%, or about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.
  • a-1 -antitrypsin of more than 50%, less than 100%, or about 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, or at least 50%, 60%, 70%, 80%, or at least 90%, or about 70%, 71 %, 72%, 73%
  • the present invention may comprise iHLCs preferably being characterized by an expression level of CYP3A7 of more than 50%, less than 100%, or about 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, or at least 50%, 60%, 70%, 80%, or at least 90%, or about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.
  • the expression level is preferably with regard to the plurality of said iHLCs, which may be used for the characterization of said iHLCs. This characterization of said iHLCs is described in Example 2.
  • target precursor stem cells preferably being selected from human or non-human induced pluripotent stem cells (iPSCs), human or non- human adult stem cells, human parthenogenetic or non-human embryonic stem cells (ESCs), a hepatocyte cell-line or primary hepatocytes.
  • target precursor stem cells may be hiPSCs.
  • the present invention may contemplate iHLCs and target precursor stem cells preferably being human cells. Further, the present invention may envisage iHLCs and target precursor stem cells being derived preferably from the same human donor.
  • "human donor” refers to a human patient used for donating his/her cells for personalized medicine. Said patient preferably donates hiPSCs being differentiated into iHLCs and hiPSCs being preferably differentiated into neural cells on iHLCs and/or on iHLC matrix.
  • the human donor preferably is a human or non-human animal, generally a mammal.
  • a mammal may be a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or preferably a human, most preferably an adult.
  • the present invention may envisage a pre-selected cell type being preferably a non-hepatocyte cell type.
  • a pre-selected cell type which is not derived from hepatocytes, is preferably a neural cell type (ectodermal origin), which may be induced by the differentiation of hiPSCs on iHLCs and/or iHLC matrix (endodermal origin).
  • ectodermal origin refers to deriving from ectoderm
  • endodermal origin refers to deriving from endoderm.
  • pre-selected means selecting in advance or choosing beforehand a cell type from a variety of cells from different germline lineages (e.g. endoderm such as pancreas, lung and liver; mesoderm such as muscle, dermis, endothelium and the circulatory system including heart and spleen; ectoderm such as brain).
  • germline lineages e.g. endoderm such as pancreas, lung and liver; mesoderm such as muscle, dermis, endothelium and the circulatory system including heart and spleen; ectoderm such as brain).
  • the present invention contemplates target cells of a pre-selected cell type preferably being neural cells.
  • Said target cells of the present invention may be mature neural cells, dopaminergic neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, serotonergic neurons, sensory neurons, astrocytes and/or oligodendrocytes.
  • said target cells of the present invention are mature neural cells, specifically dopaminergic neurons.
  • said target cells of the present invention are dopaminergic neurons.
  • the present invention provides a pre-selected cell type preferably being a neural cell type and target cells preferably being mature neural cells expressing Dcx, Map-2, Synapsin 1 , TuJ1 , NSE, Map2a, Gap43, NF, CD24, CDH2/CD325, synaptophysin, and/or CD56/NCAM; specifically dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2, cholinergic neurons expressing NGF and/or ChAT, GABAergic neurons expressing GAD67 and/or vGAT, glutamatergic neurons expressing vGLUTI , serotonergic neurons, motor neurons expressing HB9, SMN, ChAT and/or NKX6, sensory neurons expressing POU4F1 and/or peripherin, astrocytes expressing GFAP and/or Tapal, or oligodendrocytes
  • the present invention may provide a pre-selected cell type preferably being a neural cell type and target cells preferably being mature neural cells preferably expressing Map-2 and TuJ1 , most preferably Map-2.
  • the present invention may comprise target cells being dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2.
  • the present invention may provide a pre-selected cell type preferably being a neural cell type and target cells preferably being mature neural cells preferably expressing Map-2 and TuJ1 , specifically dopaminergic neurons preferably expressing Nurrl , En1 , TH and/or FoxA2, most preferably expressing Nurrl and/or En1 .
  • the present invention may contemplate a pre-selected cell type preferably being a neural cell type and target cells preferably being mature neural cells preferably expressing Map-2 and TuJ1 , specifically dopaminergic neurons preferably expressing Nurrl , En1 , TH and/or FoxA2, most preferably expressing TH.
  • the present invention may contemplate a pre-selected cell type preferably being a neural cell type and target cells preferably being mature neural cells preferably expressing Map-2 and TuJ1 , specifically dopaminergic neurons preferably expressing Nurrl , En1 , TH and/or FoxA2, most preferably expressing FoxA2.
  • the present invention may envisage target cells being mature neural cells, specifically dopaminergic neurons, which may be induced more efficiently on hiPSCs-derived iHLCs in comparison to MS5 cells.
  • the present invention may envisage target cells being mature neural cells, specifically dopaminergic neurons, which may be induced more efficiently on iHLC- derived matrix in comparison to HepG2 matrix and/or Geltrex matrix.
  • Mature neural cells preferably express Map2 (Microtubule-associated protein 2) being a cytoskeletal protein and being expressed weakly in neuronal progenitor cells (NPCs) but increases during neuron development process. In general, its expression is confined to neurons and reactive astrocytes. Mature neural cells also preferably express the neuron-specific TuJ1 (class III ⁇ -tubulin, also called beta3 Tubulin). TuJ1 may be present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal precursors.
  • Map2 Microtubule-associated protein 2
  • TuJ1 class III ⁇ -tubulin, also called beta3 Tubulin
  • TuJ1 as referred to herein may be used as pan-neuronal marker of mature neurons, which may have multiple targets (somatic, nuclear, dendritic, spine and axonal proteins) and consequently may label across all parts of the neuron.
  • pan-neuronal markers may be used for study neuronal morphology.
  • Dopaminergic neurons in particular midbrain dopaminergic neurons preferably express TH (tyrosine hydroxylase), which may be an enzyme involved in the synthesis of dopamine and norepinephrine.
  • TH tyrosine hydroxylase
  • dopaminergic neurons in particular midbrain dopaminergic neurons preferably express the transcription factor FoxA2 (Forkhead-Box-Protein A2). FoxA2 is preferably necessary for neuronal survival. Most preferably, FoxA2 is required for adult dopaminergic neurons maintenance. Nurrl may also be critical for the development and maintenance of midbrain dopaminergic (DA) neurons.
  • TH tyrosine hydroxylase
  • FoxA2 is preferably necessary for neuronal survival.
  • FoxA2 is required for adult dopaminergic neurons maintenance. Nurrl may also be critical for the development and maintenance of midbrain dopaminergic (DA) neurons.
  • En1 may lead to massive cell death of dopaminergic neurons in young adult mice, highlighting En1 as a potential gene in the molecular cascade, which may lead to progressive cell death in the neuropathology of Parkinson, being the selective and progressive degeneration of dopaminergic neurons.
  • neural induction in the differentiation of hiPSCs to midbrain dopaminergic neurons in a matrix based differentiation system may be initiated more efficiently in N2B27 1 :1 medium supplemented with the dual SMAD inhibition LDN-193189 (LDN) and SB431542 (SB) (Fig. 19 or Example 15).
  • LDN LDN-193189
  • SB SB431542
  • Neural induction or neural identity in the differentiation of hiPSCs to midbrain dopaminergic neurons may be acquired through the use of inhibitors, preferably through dual SMAD inhibition.
  • neural cell may refer to any cell that in vivo would become part of the nervous system (CNS and/or PNS) and in culture (in vitro) is obtained by methods of the present inventions.
  • Neural cells preferably being the target cells of the present invention may derive from a neural cell type.
  • Neural cells may refer to neural stem cells (NSCs), neural progenitor cells (NPCs), all mature neural cells in general, neurons including cholinergic neurons, GABAergic neurons, glutamatergic neurons, serotonergic neurons, patternable (i.e.
  • Neural cells may be divided into neural cells of early differentiation, which may include neural stem cells and/or NPCs and neural cells of late differentiation, which may include mature neural cells, specifically dopaminergic neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, serotonergic neurons, motor neurons, sensory neurons, astrocytes and/or oligodendrocytes.
  • neural refers to a neural cell and/or a neural cell type.
  • mature neural cells refers to fully developed and differentiated neural cells of late differentiation.
  • Mature neural cells may be characterized among others by different markers of late neuronal fate acquisition such as Dcx, Map2, Synapsin 1 , TuJ1 , NSE, Map2a, Gap43, NF, CD24, CDH2/CD325, synaptophysin, and/or CD56/NCAM.
  • Mature neural cells may comprise dopaminergic neurons, cholinergic neurons, GABAergic neurons, glutamatergic neurons, serotonergic neurons, sensory neurons as well as astrocytes and/or oligodendrocytes.
  • the defined neural cell type whether a mature neural cell may become a neuron specifically a dopaminergic neuron, a cholinergic neuron, a GABAergic neuron, a sensory neuron, a motor neurons, a serotonergic neuron, a glutamatergic neuron or an astrocyte or an oligodendrocyte, has not yet been determined by markers characterizing dopaminergic, cholinergic, GABAergic neurons etc. and/or markers characterizing astrocytes or oligodendrocytes.
  • the term “markers of late neuronal fate acquisition” refers to markers of neural cells of late differentiation.
  • markers of early neural fate acquisition refer to markers of neural cells of early differentiation (e.g. N-Cadherin, Pax6, Otx2, Nestin etc.).
  • the term “neuronal” refers to a neuronal cell (a neuron).
  • neurons refer to a nerve cell, which is an electrically excitable cell that processes and transmits information through electrical and chemical signals. These signals between neurons occur via synapses, specialized connections with other cells. Neurons can connect to each other to form neural networks. Neurons are the core components of the brain and spinal cord of the central nervous system (CNS), and of the ganglia of the peripheral nervous system. Neurons consist of a cell body (or soma) including the nucleus, a number of branching dendrites and an axon and various synapses at the axon terminal. Neurons may include but are not limited to dopaminergic neurons, sensory neurons, motor neurons, cholinergic neurons, serotonergic neurons, GABAergic neurons and/or glutamatergic neurons.
  • a dopaminergic (DA) neuron refers to a dopamine-producing nerve cell, wherein dopamine is a common neurotransmitter.
  • the main source of dopamine (DA) in the mammalian central nervous system results in dopaminergic neurons of the midbrain. They occur in a 'harsh' region of the brain called the substantia nigra pars compacta. This region is DA-rich and characterized by redox available neuromelanin and a high iron content.
  • a loss of dopaminergic neurons may be associated with one of the most prominent human neurological disorders, Parkinson's disease (PD).
  • Dopaminergic neurons may be characterized, among others by the expression of Nurr1 , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2.
  • a cholinergic neuron refers to a neuron that synthesizes, stores and releases acetylcholine (ACh). Such neurons may be characterized, among others by the expression of NGF and/or ChAT.
  • a GABAergic neuron refers to a neuron, which produces GABA (Gamma aminobutyric acid, a principal inhibitory neurotransmitter in the central nervous system), and is the predominant inhibitory neuron in the mammalian CNS. Approximately 60-75% of all synapses in the CNS are GABAergic. GABAergic neurons may be characterized, among others by the expression of GAD67 and/or vGAT.
  • a glutamatergic neuron is a neuron producing glutamate, which is one of the most common excitatory neurotransmitters in the central nervous system. Glutamatergic neurons may be characterized, among others, by the expression of vGLUTL
  • a serotonergic neuron refers to a neuron synthesizing the neurotransmitter serotonin (5-HT), which is found in the gastrointestinal tract, blood platelets, and the central nervous system (CNS). Serotonergic neurons may be characterized, among others by the expression of TPH and/or SERT.
  • a motor neuron refers to a neuron, whose cell body is located in the spinal cord and whose fiber (axon) projects outside the spinal cord to directly or indirectly control effector organs, mainly muscles.
  • Motor neurons' axons are efferent nerve fibers that carry signals from the spinal cord to the effectors to produce effects.
  • Types of motor neurons are alpha motor neurons, beta motor neurons, and gamma motor neurons.
  • Motor neurons may be characterized, among others by the expression of HB9, SMN, ChAT and/or NKX6.
  • a sensory neuron refers to a neuron that converts a specific type of stimulus into action potentials or graded potentials. Sensory neurons may be characterized, among others by the expression of POU4F1 and/or peripherin.
  • An astrocyte refers to a sub-type of glial cells being non-neuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the central nervous system. Astrocytes perform many functions, including provision of nutrients to the nervous tissue, maintenance of extracellular ion balance, and a role in the repair and scarring process of the brain and spinal cord. Astrocytes may express, among others, GFAP and/orTapal.
  • oligodendrocyte refers to a sub-type of glial cells. Their main functions are to provide support and insulation to axons in the central nervous system of some vertebrates by creating the myelin. Oligodendrocytes may be characterized, among others by the expression of 01 , 04, CNPase, and/or MBP.
  • the present invention envisages the pre-selected neural cell type being preferably induced by the addition of SHH activators, and/or FGF activators, and/or BMP inhibitors and/or TGFb inhibitors.
  • Sonic hedgehog is a member of the Hedgehog family of secreted signaling proteins that has a prominent role in patterning during early mammalian development.
  • SHH is expressed during early embryogenesis in a variety of tissues.
  • SHH may directly act on cells to specify neural cell fates, may regulate proliferation and survival of oligodendrocytes precursors and neural crest cells and stimulates proliferation, differentiation and axon growth (Finco et al. 2015, Annual Review of Physiology, 77:105-129).
  • the present invention may contemplate a pre-selected neural cell type being induced by SHH, which may function as an activator of dopaminergic neuron specification.
  • FGF is a fibroblast growth factor, a family consisting of 22 FGF ligands. They function as heparin-binding proteins, structurally related to signaling molecules. Since binding of the FGF to the FGF-receptor induces receptor tyrosine auto-phosphorylation, FGFs play a tremendous role in the processes of proliferation and differentiation of wide variety of cells and tissues (Zhang et al. 2008, Mol. Endocrinol, 22(1 ): 167-175).
  • the growth factor FGF8 with its two variants FGF8a and FGF8b may be expressed in the mid-hindbrain region during development.
  • FGF8 signaling is important for neural crest delamination and migration (Olsen et al. 2006, GenesDev. 20, 185-198 and Vitelli et al. 2002, Development 129, 4605-461 1 ).
  • the present invention may contemplate a pre-selected neural cell type being induced by FGF, preferably by FGF8 and/or FGF2, which may function as an activator for dopaminergic neuron specification.
  • DA dopaminergic
  • SHH and FGF preferably SHH and FGF8 for their development
  • the present invention may contemplate a pre-selected neural cell type being induced by SHH activators and/or FGF activators, preferably by SHH activators and/or FGF8 activators and/or FGF2 activators.
  • BMP is a bone morphogenetic protein that refers to a multi-functional growth factor and belongs to the transforming growth factor beta (TGFb) superfamily.
  • TGFb transforming growth factor beta
  • the TGF beta superfamily of ligands include besides BMPs, GDFs, Activin, Nodal and TGFbs.
  • BMPs transforming growth factor beta
  • GDFs transforming growth factor beta
  • Activin Nodal
  • TGFbs transforming growth factor beta
  • signaling begins.
  • Each class of ligand binds to a specific type II receptor, thus for a BMP ligand a type II receptor may refers to BMPR2 and for a TGFb ligand a type II receptor may refer to TGFbRII.
  • the type II receptor is a serine/threonine receptor kinase, which catalyzes the phosphorylation of the Type I receptor.
  • a type I receptor may refer to ALK2 and ALK3 for a BMP signaling pathway (Alberts et al. 2002, Molecular Biology of the Cell. New York, NY: Garland Scienc).
  • BMP signaling is important in heart, neural and cartilage development and also plays a crucial role in postnatal bone formation (Chen et al. 2004, Growth Factors, 22(4):233- 41 ).
  • BMP inhibitors including chordin (Sasai, et al. 1994, Cell 79(5):779), follistatin (Hemmati-Brivanlou, et al. 1994, Cell 77(2):283), and noggin (Smith, et al. 1992, Cell 70(5):829) as the critical neural inducing factors.
  • LDN-193189 may also be a selective BMP signaling inhibitor, which may inhibit the transcriptional activity of the BMP type I receptors ALK2 and ALK3 and may prevent phosphorylation of their downstream targets SMAD1 , SMAD5 and SMAD8.
  • LDN-193189 may have little effect on ALK4, ALK5, and ALK7, which may be inhibited by a specific TGFb inhibitor.
  • the LDN inhibitor of BMP type I receptors ALK2 and ALK3 may be used for the induction of neural fate in the present invention.
  • the present invention may contemplate a pre-selected neural cell type being induced by BMP inhibitors, preferably by the ALK2/3 inhibitor LDN-193189.
  • TGFb includes transforming growth factor-beta as well as functional equivalents, derivatives and analogs thereof.
  • the TGF-beta isoforms are a family of multifunctional, disulfide-linked dimeric polypeptides that affect activity, proliferation and differentiation of various cells types.
  • TGF-beta is a polypeptide produced in a latent propeptide form having, at this time, no identified biological activity. To be rendered active and, the propeptide form of TGF-beta must be cleaved to yield active TGFb.
  • the specific inhibitor which may block the TGFb pathway is preferably the potent inhibitor SB431542 (Tocris).
  • SB-431542 is a small molecule inhibitor that was identified as an inhibitor of Activin receptor-like kinase ALK5, which is the TGFb type I receptor. Besides inhibiting ALK5 it also inhibits the Activin type I receptor ALK4 and the Nodal type I receptor ALK7 (Gareth et al. 2002, Molecular Pharmacology, 62 (1 ) 65-74).
  • SB431542 is a drug shown to enhance neural induction in an embryoid body (EB) based hESC neural induction protocol (Smith, et al. 2008, Dev Biol 313(1 ): 107) and which also blocks the Activin and Nodal signaling pathway.
  • EB embryoid body
  • ALK4, ALK5, and ALK7 prevents the phorphorylation of their downstream targets SMAD2 and SMAD3.
  • This specific inhibitor of TGFb may be among others used for the induction of neural fate in the present invention.
  • the present invention may contemplate a pre-selected neural cell type being induced by TGFb inhibitors, preferably by the ALK4/5/7 inhibitor SB431542.
  • the present invention may provide a pre-selected neural cell type being induced by BMP inhibitors and/or TGFb inhibitors.
  • a pre-selected neural cell type may be induced by LDN-193189 (LDN) and SB431542 (SB), both being preferably referred to dual SMAD inhibition.
  • dual SMAD inhibition as referred to herein may be determined by LDN-193189 (LDN) and SB431542 (SB).
  • LDN-193189 (LDN) and SB431542 (SB) may also refer to as indirect SMAD protein inhibitors.
  • LDN-193189 (LDN) and SB431542 (SB) may also refer to indirect inhibitors of SMAD proteins 1 , 2, 3, 5, and 8. Dual SMAD inhibition may therefore be essential for inducing neural fate.
  • the present invention may envisage a pre-selected cell type being the neural cell type being induced by dual SMAD inhibition.
  • SMAD inhibition refers to the direct and/or indirect inhibition of the Small Mothers against Decapentaplegic proteins.
  • SMAD proteins are downstream of TGFb superfamily ligands, and when their specific receptors are activated, they stimulate the phosphorylation of the receptor-regulated SMADs.
  • Receptor-regulated-SMADs may refer to SMAD1 , SMAD2, SMAD3, SMAD5 and SMAD 8, with SMADs 2 and 3 specifically being activated by Activin/Nodal and TGF-beta type I receptors.
  • SMADs 1 , 5, and 8 specifically being activated by BMP type I receptors.
  • Dual SMAD inhibition may take a confluent culture of hiPSCs and rapidly may differentiate them into early neurectoderm. This rapid differentiation may be caused by blocking the two signaling pathways that utilize SMADs for transduction: BMP and TGFb.
  • dual SMAD inhibition may refer to the inhibitors LDN 193189 and SB431542, which may indirectly inhibit SMAD proteins.
  • the present invention may also contemplate a composition comprising (a) immature hepatocyte-like cells (iHLCs) and/or (b) an iHLC-derived extracellular matrix (ECM) as a culture system and (c) target precursor stem cells differentiating into target cells of a pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • ECM extracellular matrix
  • Contemplated by the invention is a composition described herein, wherein said target precursor stem cells are preferably selected from human or non-human iPSCs, human or non- human adult stem cells, and human parthenogenetic or non-human ESCs, a hepatocyte cell- line or primary hepatocytes.
  • the present invention may comprise a composition, wherein said target precursor stem cells may be hiPSCs.
  • the present invention may also comprise a composition described herein, wherein said target cells of the composition are preferably mature neural cells expressing Dcx, Map-2, Synapsin 1 , TuJ1 , NSE, Map2a, Gap43, NF, CD24, CDH2/CD325, synaptophysin, and/or CD56/NCAM, specifically are dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2, cholinergic neurons expressing NGF and/or ChAT, GABAergic neurons expressing GAD67 and/or vGAT, glutamatergic neurons expressing vGLUTI , serotonergic neurons, motor neurons expressing HB9, SMN, ChAT and/or NKX6 as well as sensory neurons expressing POU4F1 and/or peripherin.
  • astrocytes expressing GFAP and/or Tapal or oligodendrocytes expressing 01 , 04, CNPase, and/or MBP.
  • said target cells of the composition contemplated by the invention are dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2.
  • the present invention may also envisage a composition described herein, comprising said target precursor stem cells and/or target cells of a pre-selected cell type in a 2D- or 3D-cell culture.
  • the present invention may envisage a composition comprising said target precursor stem cells and/or target cells of a pre-selected cell type in 3D-cell culture.
  • the cultivation of cells on a flat plastic surface leads to an artificial, two- dimensional cell layer.
  • Normal cells in the human body develop in a three-dimensional environment, surrounded by other cells, membranes and adhesion proteins etc.
  • the present invention provides a 3D- cell culture system, offering an in vivo morphology and a particular environment for more realistic cell biology.
  • the present invention may also provide a method for preparing an iHLC-derived cell-free ECM, comprising (a) cultivating iHLCs in vitro under conditions allowing for formation of an iHLC-derived ECM, (b) decellularizing the iHLC-derived ECM and (c) obtaining the iHLC- derived cell-free ECM.
  • cultivating may refer to letting cells grow and differentiate under certain conditions that may also enable or allow the process of forming an ECM, preferably an iHLC-derived ECM.
  • decellularizing in this context, may include washing iHLCs once with PBS and then adding the decellularization buffer to the cells, which may contain 20mM ammonium hydroxide, 0.5% Triton X-100 in PBS and which is preferably steril-filtered.
  • the decellularization buffer may isolate the ECM derived from said iHLCs from its inhabiting cells, said iHLCs (Example 3).
  • the present invention may also comprise an immature hepatocyte-like cell (iHLC) derived extracellular matrix preferably obtained or obtainable by the method as mentioned above.
  • iHLC immature hepatocyte-like cell
  • the differentiation of hiPSC on iHLC matrix is the most efficient culture system for dopaminergic differentiation compared to HepG2 matrix.
  • iHLC immature hepatocyte-like cell
  • HepG2 cells and/or HepG2 conditioned medium nor HepG2 matrix may be used as an in vitro culture system for the differentiation of target precursor stem cells into target cells of a pre-selected cell type.
  • HepG2 cells or HepG2 conditioned medium or HepG2 matrix may not induce neural fate in comparison to iHLCs and/or an ECM derived from said iHLCs of the present invention.
  • the present invention may envisage an iHLC-derived ECM obtained or obtainable by the method as mentioned above for dopaminergic neuron differentiation in comparison to a HepG2 matrix.
  • a HepG2 matrix may refer to the extracellular matrix derived from (produced from) HepG2 cells being a human hepatocellular carcinoma cell line.
  • the present invention may comprise an iHLC-derived extracellular matrix preferably characterized by the expression of Lamal , Lama5, Larmbl , Lamb2, Lamb3, Larnd , Lamc2, Perlecan, Thbsl , Nidi , Vcan, Ltbpl Col5a1 , Agrn, Fbn2, Gdf6, Haplnl , Igfbp2, Igfbp5, Mdk, Vtn, Col3A1 , Col4A1 , Fbln2, Tnc, and SfrpL
  • An iHLC matrix may provide a specific combination of factors or ECM components for neural induction, specifically for dopaminergic neuron induction.
  • This specific combination of factors or ECM components may refer to the specific signature of the iHLC matrix.
  • signature of the iHLC matrix may refer to specific ECM proteins (candidate proteins) being expressed in the iHLC matrix, which may give the matrix the ability to induce neural fate, preferably dopaminergic neurons.
  • Lama5 Lamal , Lama5, Larmbl , Lamb2, Lamb3, Larnd , Lamc2, Perlecan (Hspg2), Thbsl , Nidi , Vcan, Ltbpl Col5a1 , Agrn, Fbn2, Gdf6, Haplnl , Igfbp2, Igfbp5, Mdk, Vtn, Col3A1 , Col4A1 , Fbln2, Tnc, and Sfrpl , but not limited to may be considered as those candidate proteins (ECM proteins), which may represent the specific signature of the iHLC matrix.
  • ECM proteins candidate proteins
  • Lamal , Lama5, Larmbl , Larnd , Ltbpl , Thbsl , Nidi , Agrn, Tnc, Fblnl , Vtn, Fbn2, Haplnl , Igfbp2 and Igfbp5 may refer to ECM glycoproteins, whereas Perlecan and/or Vcan are preferably proteoglycans and Col4a1 and Col3a1 may refer to collagens. ECM glycoproteins, collagens and proteoglycans may refer to core matrisome proteins (proteins of the core matrisome).
  • Mdk, Sfrpl , Gdf6 may refer to a matrisome-associated protein, which may comprise ECM-affiliated proteins, ECM regulators and secreted factors, in particular Mdk, Sfrpl and Gdf6 may refer to a secreted factor.
  • the present invention may further comprise an iHLC-derived ECM being preferably characterized by Larmbl , Larnd and/or Lamal in comparison to HepG2 cells.
  • the present invention may further comprise an iHLC-derived ECM being preferably characterized by Agrn, Fbn2, Gdf6, Haplnl , Igfbp2, Igfbp5, Larmbl and Ltbpl in comparison to HepG2 cells, hiPSCs and mesenchymal stem cell (MSC).
  • Fbn2, Igfbp2, Igfbp5 and Ltbpl are involved in TGF3- signalling, Gdf6 in BMP-signalling and Agrn, Haplnl and Larmbl in CNS development.
  • the present invention may further comprise an iHLC-derived ECM being preferably characterized by Mdk and Vtn in comparison to MSC and hiPSCs. Mdk and Vtn are associated with CNS development.
  • the present invention may further comprise an iHLC-derived ECM being preferably characterized by Col3a1 , Col4a1 , Fblnl , Thbsl , and Tnc in comparison to hiPSCs and HepG2 cells.
  • the present invention may further comprise an iHLC-derived ECM being preferably characterized by Sfrpl in comparison to MSC and HepG2 cells. Sfrpl is involved in dopamine neuron development.
  • the present invention may also comprise an iHLC-derived ECM being preferably characterized by FGF2 in comparison to HepG2 cells. FGF2 may also refer to a matrisome-associated protein of an extracellular matrix, in particular to a secreted factor as mentioned above.
  • the present invention may provide an iHLC-derived ECM supporting neural induction characterized by Lama5, Larmbl , Lamb2, Larnd , Perlecan, Thbsl , Nid 1 , Vcan, Agrn, Haplnl , Fbn2, Igfbp2, Igfbp5, Mdk, Vtn, Col3a1 , Col4a1 , Tnc, Fblnl .
  • the present invention may comprise an iHLC-derived ECM supporting dopaminergic neuron induction characterized by Lamal , Lamb3, Lamc2, Ltbpl , Col5a1 , Sfrpl , Tnc.
  • the iHLC-derived ECM contemplated by the invention is preferably further (a) highly concentrated, (b) growth factor reduced, (c) phenol-red free and/or (d) suitable for human embryonic stem cell (hESC) cultivation.
  • the term “highly concentrated” may refer to higher protein concentration (HC), which provides greater matrix stiffness and scaffold integrity.
  • a preferred protein concentration may comprise more than 10 mg/mL, less than 30 mg/mL, or 10-30 mg/mL, 15-25 mg/mL, 18-21 mg/mL, or at least 10, 15, 18, 19, 20, or at least 21 mg/mL. Preferably, 18 mg/mL to 21 mg/mL.
  • growth factor reduced (GFR) may refer to applications, where more highly defined basement membrane preparation may be desired.
  • a preferred protein concentration may comprise more than 5 mg/mL, less than 20 mg/mL, or 5-20 mg/mL, 7-15 mg/mL, 8-12 mg/mL, or at least 5 mg/mL, 8, 9, 10, 1 1 , or at least 12 mg/mL. Preferably, 8 mg/mL to 12 mg/mL.
  • the term "phenol-red free” may refer to assays that require color detection (e.g., colorimetric, fluorescence).
  • a phenol red free medium may not include Phenol Red pH- indicator.
  • a preferred protein concentration of a phenol-red free medium may comprise more than 5 mg/mL, less than 20 mg/mL, or 5-20 mg/mL, 7-15 mg/mL, 8-12 mg/mL, or at least 5 mg/mL, 8, 9, 10, 1 1 , or at least 12 mg/mL. Preferably, 8 mg/mL to 12 mg/mL.
  • the iHLC-derived ECM of the present invention may comprise certain candidate proteins as mentioned above, it may also comprise some characteristics of the standard Corning Matrigel matrix of the literature, which may also be highly concentrated, growth factor reduced and/or phenol-red free.
  • a Corning Matrigel matrix may refer to a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, including Laminin (a major component of said Matrigel), Collagen IV, heparin sulfate proteoglycans, entactin/nidogen, and a number of growth factors.
  • said ECM derived from said iHLCs may provide the reproducibility and consistency essential for human embryonic and/or induced pluripotent feeder-free stem cell culture.
  • the present invention may envisage an ECM derived from said iHLCs comprising a perfect combination of specific ECM components for the induction of neural fate and standard formulation of ECMs known from the literature.
  • the present invention may encompass a device comprising immature hepatocyte-like cells (iHLCs) and/or an extracellular matrix (ECM) derived from said iHLC as a culture system.
  • the device is a vial or a cell-culture dish.
  • the vials of the present invention may comprise centrifuge tubes comprising different sizes.
  • the present invention may include centrifuge tubes (e.g. polypropylene centrifuge tubes) comprising 10, 15, 20 25, 30, 35, 40 or 50 ml working volume.
  • the present invention may envisage cell-culture dishes comprising petri dishes of different sizes.
  • the present invention may include petri-dishes comprising 3, 4, 5, 6, 7, 8, 9, or 10 ml working volume.
  • the present invention may envisage cell-culture dishes comprising flasks of different sizes.
  • the present invention may include flasks comprising 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or greater than 300 ml working volume.
  • the vials and the cell-culture dishes may include the ECM preferably derived from hiPSC derived iHLCs, which may provide a 3D-cell culture system, offering an in vivo morphology and a particular environment for more realistic cell biology
  • the present invention may envisage a device comprising target precursor stem cells of the present invention or target cells of a pre-selected cell type of the present invention.
  • Said device may comprise target precursor stem cells, wherein said target precursor stem cells are preferably selected from human or non-human iPSCs, human or non-human adult stem cells, and human parthenogenetic or non-human ESCs, a hepatocyte cell-line or primary hepatocytes.
  • the device may comprise hiPSCs as target precursor stem cells.
  • the device may comprise target cells of a pre-selected cell type, wherein said pre-selected cell type is a non-hepatocyte cell type, preferably a neural cell type.
  • the device may comprise target cells of a pre-selected cell type, wherein said target cells are mature neural cells expressing Dcx, Map-2, Synapsin 1 , TuJ1 , NSE, Map2a, Gap43, NF, CD24, CDH2/CD325, synaptophysin, and/or CD56/NCAM; specifically dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2, cholinergic neurons expressing NGF and/or ChAT, GABAergic neurons expressing GAD67 and/or vGAT, glutamatergic neurons expressing vGLUTI , serotonergic neurons, motor neurons expressing HB9, SMN, ChAT and/or NKX6, sensory neurons expressing POU4F1 and/or peripherin, astrocytes expressing GFAP and/or Tapal, or oligodendrocytes expressing 01 , 04, C
  • the device comprises target cells of a pre-selected cell type, wherein said target cells preferably are dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2.
  • target cells preferably are dopaminergic neurons expressing Nurrl , En1 , TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2.
  • the invention may also contemplate an in vitro method for obtaining target cells of a preselected cell type, comprising (a) providing immature hepatocyte-like cells (iHLCs) and/or an iHLC-derived ECM from the present invention, (b) adding target precursor stem cells from the present invention in a suitable cell culture medium and allowing their differentiation into the target cells of a pre-selected cell type defined by the invention; (c) obtaining the target cells of the pre-selected cell type.
  • iHLCs immature hepatocyte-like cells
  • iHLC-derived ECM from the present invention
  • target precursor stem cells from the present invention in a suitable cell culture medium and allowing their differentiation into the target cells of a pre-selected cell type defined by the invention
  • obtaining the target cells of the pre-selected cell type comprising (a) providing immature hepatocyte-like cells (iHLCs) and/or an iHLC-derived ECM from the present invention.
  • providing iHLC and/or iHLC-derived ECM may refer to the differentiation of target precursor stem cells into iHLCs (Example 1 ) and the obtainment of an extracellular matrix derived from said iHLCs, which may be decellularized with a specific decellularization buffer containing 20mM ammonium hydroxide, 0.5% Triton X-100 (Example 3).
  • a suitable cell culture medium may refer to the medium, which may be used for the differentiation of target precursor stem cells on iHLCs (Example 4) and/or on hiPSCs-derived iHLC matrix (Example 6 and Example 15) into the target cells of a pre-selected cell type.
  • neural induction in a rosette based differentiation e.g.
  • differentiation of hiPSCs on iHLCs may be achieved with KSR medium containing Knock out DMEM, 15% Knock out serum replacement (KOSR), 2 mM L-glutamine, non-essential amino acids and 10 ⁇ ⁇ - mercaptoethanol supplemented with 1 ⁇ dorsomorphine (Tocris) and 10 ⁇ SB431542 (Miltenyi) and 100 ng ml "1 SHH C24II (Miltenyi) and 100 ng ml "1 FGF8 (Miltenyi), as it is described in Example 4.
  • Neural induction of midbrain dopaminergic neurons (mDA) in a floor- plate based differentiation e.g.
  • differentiation of hiPSCs on iHLC matrix may be achieved with KSR medium supplemented with 100 nM LDN193189 (Stemgent), 10 ⁇ SB431542 (Miltenyi), 100 ng ml "1 SHH C24II (Miltenyi), 2 ⁇ Purmorphamine (Stemgent), 100 ng ml "1 FGF8 (Miltenyi) and 3 ⁇ CHIR99021 (Miltenyi), as it is described in Example 6.
  • neural induction of midbrain dopaminergic neurons (mDA) of hiPSCs on iHLC matrix may also be achieved with N2B27 1 :1 medium supplemented with 100 nM LDN193189 and 10 ⁇ SB431542, with 100 ng ml "1 SHH C24II (Miltenyi) and 100 ng ml "1 FGF8 (Miltenyi), as it is described in Example 15.
  • the present invention may further provide the in vitro method, wherein said target cells are mature neural cells optionally expressing Dcx, Map-2, Synapsin 1 , TuJ1 , NSE, Map2a, Gap43, NF, CD24, CDH2/CD325, synaptophysin, and/or CD56/NCAM, specifically dopaminergic neurons expressing TH, AaDC, Dat, Otx-2, FoxA2, LMX1A and/or VMAT2, cholinergic neurons expressing NGF and/or ChAT, GABAergic neurons expressing GAD67 and/or vGAT, glutamatergic neurons expressing vGLUTI , serotonergic neurons, motor neurons expressing HB9, SMN, ChAT and/or NKX6, sensory neurons expressing POU4F1 and/or peripherin, astrocytes expressing GFAP and/or Tapal, or oligodendrocytes expressing 01 , 04, CNPase, and/or MBP
  • the present invention may contemplate a target cell of a pre-selected cell type obtained or obtainable by the in vitro method mentioned above for use in a method of cell grafting in a patient in need thereof.
  • Cell grafting or neural transplantation involves a process for transplanting genetically modified donor cells, preferably neural cells from the present invention, into the central nervous system (CNS) to treat specific diseases.
  • Conditions for successful transplantation may include: 1 ) viability of the implant; 2) retention of the graft at the site of transplantation; and 3) minimum amount of pathological reaction at the site of transplantation.
  • the patient may be a non-human animal, a mammal, including rabbits, rats, rabbits, pigs, mice, hamsters, dogs, cats, cows, goats, sheeps, horses, monkeys, apes and/or humans.
  • the patient may be any subject as defined herein, preferably a human subject. In one embodiment, the subject is an adult.
  • the present invention may contemplate a target cell preferably characterized by the increased expression of FoxA2, TH, Map2, Nestin, Pax6, Otx2, En1 , LmxIA and/or Nurrl in comparison to a target cell obtained by cultivation in MS5 or Geltrex.
  • said target cell of the present invention may be used in a method for treating a neurodegenerative disease, including without limitation Alzheimer's Disease, Parkinson, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Muscular Dystrophy, preferably Parkinson. Additionally, said target cell of the present invention may be used in a method for treating a neurodegenerative disease, including without limitation Alzheimer's Disease, Parkinson, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Muscular Dystrophy in a subject.
  • a neurodegenerative disease including without limitation Alzheimer's Disease, Parkinson, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Muscular Dystrophy in a subject.
  • the subject may be a non-human animal, a mammal, including rabbits, rats, rabbits, pigs, mice, hamsters, dogs, cats, cows, goats, sheeps, horses, monkeys, apes and/or humans, preferably a human.
  • the subject is an adult.
  • the present invention may comprise target cells, specifically dopaminergic neurons expressing Nurrl and/or En1 for use in a method for treating a neurodegenerative disease, including without limitation Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Muscular Dystrophy, preferably Parkinson's Disease.
  • a neurodegenerative disease including without limitation Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Muscular Dystrophy, preferably Parkinson's Disease.
  • Neurodegenerative disease may refer to a wide range of diseases and/or disorders of the central and peripheral nervous system, such as Parkinson's disease, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), denervation atrophy, otosclerosis, stroke, dementia, multiple sclerosis, Huntington's disease, encephalopathy associated with acquired immunodeficiency disease (AIDS), and other diseases associated with neuronal cell toxicity and cell death.
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • otosclerosis otosclerosis
  • stroke dementia
  • dementia dementia
  • multiple sclerosis Huntington's disease
  • AIDS acquired immunodeficiency disease
  • AIDS acquired immunodeficiency disease
  • in vitro refers to studies that are conducted using components of an organism that have been isolated from their usual biological surroundings, such as cells, or biological molecules.
  • cells can be studied in artificial culture medium, proteins can be examined in solutions.
  • test-tube experiments these studies in biology, medicine and its sub-disciplines are traditionally done in test tubes, flasks, petri dishes, etc.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment, such as embryonic development, cell differentiation, neural tube formation, etc.
  • culture system refers to the use of different media containing different ingredients and supplements for differentiating cells in particular.
  • cell culture refers to any in vitro culture of cells. Included within this term may be continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • cultivation refers to the process of cultivating cells, cell strains or lines in the laboratory preferably on matrices or feeder cells that the cells are able to grow, differentiate, induce certain markers and/or mature to fully developed cells.
  • derived from may be used interchangeably with the term “originated from” or “obtained from”.
  • an extracellular matrix may be obtained preferably from iHLCs or iHLCs originating preferably from hiPSCs.
  • the term “substantially” may be used interchangeably with the term “significantly” or “extremely”.
  • the ECM of the present invention being substantially cell-free means that the ECM is extremely isolated of its inhabiting cells, preferably of said iHLCs of the present invention.
  • the term "fate” in general refers to a cell with a genetically determined lineage whose progeny cells are capable of becoming a variety of cell types or a few specific cell types depending upon in vivo or in vitro culture conditions.
  • a cell's predetermined fate is determined by it's environment to be destined for a particular differentiation pathway such that a cell becomes one cell type instead of another cell type.
  • "neural fate” may refer to hiPSCs which may become a neural cell instead of a muscle cell or a skin cell after differentiation of hiPSCs on hiPSCs-derived iHLCs and/or iHLC matrix.
  • an “activator” may refer to a protein (transcription factor) that increases gene transcription of a certain gene/set of genes or which activates a signaling process or a differentiation process of cells.
  • SHH and/or FGF activators as used herein may refer to the proteins SHH and/or FGF, preferably to SHH and/or FGF8 that activate neural differentiation of hiPSCs into midbrain dopaminergic neurons.
  • an “inhibitor” may refer to a protein that decreases gene transcription of a certain gene/set of genes or which inhibits a signaling cascade.
  • BMP and/or TGFb inhibitors may be inhibitors that may inhibit BMP and/or TGFb signaling, which may be acquired for neural identity.
  • a BMP inhibitor may be LDN
  • a TGFb inhibitor may be SB.
  • the term "increased" preferably means that an expression level of a protein is higher for specific cells which may be cultivated under certain conditions of the present invention than an expression level of a protein for cells being cultivated under conditions known in the prior art.
  • the term "plurality of iHLCs” may refer to a specific amount of iHLCs in a well of a 48 well plate (about 100.000 cells in a well of a 48 well plate) or in a well of a 96 well plate (about 30.000 cells in a well of a 96 well plate), which is used for the characterization of said iHLCs in immunocytochemistry, preferably in immunocytochemistry pictures of about 200-500 cells are taken and the expression of iHLCs markers is examined. The characterization of said iHLCs is described in Example 2.
  • feeder cells may refer to cells of one tissue type that are co-cultured with cells of another (second) tissue type, to provide an environment in which the cells of the second tissue type can grow.
  • the feeder cells are optionally from a different species as the cells they are supporting.
  • the feeder cells may be MS5 cells, hiPSCs-derived iHLCs, HepG2 cells, hiPSC derived cardiomyocytes or hiPSC derived mesenchymal progenitor cells (MPCs), preferably hiPSCs-derived iHLCs.
  • the cells, which are co-cultured with those feeders may be human or non-human iPSCs, human or non-human adult stem cells, human parthenogenetic or non-human ESCs, preferably hiPSCs.
  • feeder free or “feeder-free matrix-based system” refers to the differentiation of cells of a tissue type not being co-cultured with other cells of another tissue type that provide an environment in which the cells being differentiated are able to grow.
  • Feeder-free matrices may be Geltrex or Matrigel matrices.
  • the iHLC matrix of the present invention may also be considered as feeder free.
  • Feeder free may be used interchangeably with the term cell-free or decellularized.
  • Feeder-free cells, preferably hiPSCs do not grow as round colonies with defined edges likes cells on feeder layers. Commonly, they grow as a monolayer.
  • feeder-free matrices may be preferred in the present invention.
  • the term “less than” or in turn “greater than” includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively.
  • Example 1 Differentiation of hiPSCs to immature hepatocyte like cells (iHLCs).
  • hiPSC need to be differentiated into iHLCs for further differentiation of hiPSCs on iHLCs (Example 4) or on an iHLC-matrix (Example 6 or Example 15) into neural fate.
  • hiPSCs were dissociated by accutase and seeded on geltrex coated dishes in mTeSRI supplemented with 10 ⁇ Rock inhibitor Y-27632.
  • Definitive endoderm differentiation was initiated by changing the medium to RPMI containing 1 % B27 w/o insulin supplemented with 100 ng ml "1 FGF2 (Miltenyi) and 100 ng ml "1 Activin A (Miltenyi).
  • hepatocyte differentiation medium was changed at day 8 to DMEM/F12 1 :1 containing 10% Knock out serum replacement (KOSR), 2 mM L-glutamine, 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin, nonessential amino acids supplemented with additional D-glucose to a final concentration 4.5 mg L " 100 ng ml "1 human HGF (Miltenyi) and 1 %DMSO (Sigma).
  • human HGF and DMSO were removed and medium was supplemented with 1 ⁇ dexamethasone (Sigma) for 3 days.
  • hepatic maturation cells were cultured at day 19 in William's E medium supplemented with 10% FBS (fetalclone II, Hyclone, Thermo Scientific), 2 mM L-glutamine, 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin, 0.023 IE ml "1 human insulin (Sigma), 4.7 ⁇ g ml “1 hydrocortisone (Sigma), 80 ⁇ g ml "1 gentamicin (Sigma) and 1 .8 % DMSO (Sigma) until the desired maturation state (Fig. 3A).
  • FBS fetalclone II, Hyclone, Thermo Scientific
  • 2 mM L-glutamine 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin, 0.023 IE ml “1 human insulin (Sigma), 4.7 ⁇ g ml “1 hydrocortisone (Sigma), 80 ⁇ g
  • iHLCs were seeded at day 22 on Collagen I (10 ⁇ g cm 2 ) coated dishes (50.000 cells/cm 2 ) in William's E medium supplemented with 10% FBS (fetalclone II, Hyclone, Thermo Scientific), 2 mM L-glutamine, 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin, 0.023 IE ml "1 human insulin (Sigma), 4.7 ⁇ g ml "1 hydrocortisone (Sigma), 80 ⁇ g ml "1 gentamicin (Sigma) and 1 .8 % DMSO (Sigma). Cells were used on the following day for mDA co-culture based differentiation experiments and after 3 days for matrix based mDA differentiation experiments (Fig. 1 ).
  • Example 2 Markers of immature hepatocytes expressed in iHLCs differentiated from hiPSCs.
  • iHLCs were characterized by the expression of marker of immature hepatocytes.
  • hiPSCs were obtained and differentiated into iHLC as described in Example 1.
  • iHLCs were subjected to immunocytochemistry using antibodies against markers of immature and mature hepatocytes HNF4a, Albumin (green) and Nkx2.5 and a-fetoprotein (AFP) (red) (Fig. 2A).
  • iHLCs were characterized and compared to HepG2 cells.
  • HepG2 cells were obtained in parallel and mRNA was isolated from HepG2 as well as from iHLCs.
  • Real-time PCR was then performed for other markers of immature hepatocytes such as OATP2B1 , AFP, HNF4a, CYP3A7 as well as for mature hepatocyte markers such as albumin, a-1 -antitrypsin, CYP3A4 and CYP7A1 (Fig. 2B and C).
  • Beta-actin was used as a control in Fig. 2B.
  • iHLCs expressed all markers of immature hepatocytes except CYP7A1 which was expressed in mature HepG2 cells, a human hepatocyte cell line.
  • Example 3 Decellularization of hiPSCs-derived iHLCs and/or HepG2 matrix.
  • iHLCs and/or HepG2 cells were seeded 3 days before decellularization in the appropriate medium at a density of 5 x 10 4 cells cm 2 .
  • decellularization buffer (20 mM ammonium hydroxide, 0.5% Triton X-100 in PBS, steril filtered 0,22 ⁇ ) was preheated to 37 °C.
  • hiPSCs-derived iHLCs were washed once with PBS and decellularization buffer was added to the cells, following incubation for 5 min at RT. Buffer was carefully taken of and cells were washed 3 times with PBS. Matrix was stored in PBS until hiPSCs were seeded.
  • Example 4 Differentiation of hiPSCs on iPSC-derived iHLCs, according to Kriks et al. (201 1 ), rosette based differentiation.
  • hiPSCs-derived iHLCs were treated with 10 ⁇ g ml "1 mitomycin C in final hepatocyte maturation medium (see above) for 1 .5 hours at 37 °C. Cells were washed twice with PBS and stored in hepatocyte medium until seeding of hiPSCs. On day 0 of differentiation hiPSCs were seeded in clumps at a density of 1500 cells per cm 2 on mitomycin C treated hepatocytes in KSR containing Knock out DMEM, 15% Knock out serum replacement, 2 mM L-glutamine, nonessential amino acids and 10 ⁇ ⁇ -mercaptoethanol.
  • Cells were co-cultured with mitomycin treated hepatocytes for 18 days in KSR supplemented with 1 ⁇ dorsomorphine (Tocris) and 10 ⁇ SB431542 (Miltenyi) from day 2 to day 6 and 100 ng ml "1 SHH C24II (Miltenyi) and 100 ng ml "1 FGF8 (Miltenyi) from day 6 to day 18 of differentiation.
  • Tocris dorsomorphine
  • SB431542 SB431542
  • neural rosettes were mechanically isolated and plated on dishes pre-coated with polyornithine (15 ⁇ g ml "1 ) and laminin (4 ⁇ g ml “1 ) in Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2 mM L- glutamine supplemented with 100 ng ml "1 SHH C24II, 100 ng ml "1 FGF8, 20 ng ml "1 BDNF (Miltenyi), 0.2 mM ascorbic acid (Sigma).
  • cells were dissociated by accutase and seeded in droplets at a density of 20.000 cells per 20 ⁇ on dishes pre-coated with polyornithine (15 ml "1 ) laminin (4 ml “1 ) and fibronectin (1 ml “1 ) (P/O+LAM/FN) in N2/B27 supplemented with100 ng ml "1 SHH C24II , 100 ng ml "1 FGF8, 20 ng ml "1 BDNF, 0.2 mM ascorbic acid.
  • Example 5 Differentiation of hiPSCs on irradiated MS5 cells (as a control), according to Kriks et al. (201 1 ); rosette based differentiation.
  • MS5 Differentiation of hiPSCs on the mouse stromal cell line MS5 was performed as described above (differentiation of iPSCs on iPSC-derived iHLCs), except from co-culture of iPSCs on irradiated MS5 for 1 1 days instead of 18 days. MS5 cells function as a control for the differentiation of hiPSC into neural fate.
  • irradiated MS5 cells were plated at a density of 50.000 cells per cm 2 in alpha- MEM containing 10% FBS and 2 mM L-glutamine.
  • hiPSCs were seeded in clumps at a density of 1500 cells per cm 2 on MS5 cells in KSR containing Knock out DMEM, 15% Knock out serum replacement, 2 mM L-glutamine, non-essential amino acids and 10 ⁇ ⁇ -mercaptoethanol, supplemented with 1 ⁇ dorsomorphine (Tocris) and 10 ⁇ SB431542 (Miltenyi) from day 2 to day 6 and 100 ng ml "1 SHH C24I I (Miltenyi) and 100 ng ml "1 FGF8 (Miltenyi) from day 6 to day 1 1 of differentiation.
  • Tocris dorsomorphine
  • neural rosettes were mechanically isolated and plated on dishes pre-coated with polyornithine (15 ⁇ g ml "1 ) and laminin (4 ⁇ g ml “1 ) in Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2 mM L-glutamine supplemented with 100 ng ml "1 SHH C24I I, 100 ng ml "1 FGF8, 20 ng ml "1 BDNF (Miltenyi), 0.2 mM ascorbic acid (Sigma).
  • Example 6 Differentiation of hiPSCs on human iPSC-derived hepatocyte matrix and Geltrex, according to Kriks et al. (201 1 ); floor-plate based differentiation.
  • Human iPS cells were dissociated by accutase and seeded at a cell density of 5 x 10 4 cells per cm 2 on human iPS-derived hepatocyte matrix or Geltrex (GeltrexTM LDEV-Free Reduced Growth Factor Basement Membrane Matrix, Thermo Fisher Scientific) coated dishes in mTeSRI supplemented with 10 ⁇ Rock inhibitor.
  • Geltrex GeltrexTM LDEV-Free Reduced Growth Factor Basement Membrane Matrix, Thermo Fisher Scientific
  • KSR medium was gradually shifted to N2/B27 medium containing Neurobasal, 1 % N2, 1 % B27 w/o Vitamin A and 2 mM L-glutamine, starting from day 5 on (Day 5: 75% KSR, 25% N2/B27; Day 7: 50% KSR, 50% N2/B27; Day 9 25% KSR, 75% N2/B27) with exposure to the following factors from day 5 to day 6 to 100 nM LDN193189, 10 ⁇ SB431542, 100 ng ml "1 SHH C24II, 2 ⁇ Purmorphamine, 100 ng ml "1 FGF8, 3 ⁇ CHIR99021 and day 7 until day 10 to 100 nM LDN193189, 3 ⁇ CHIR99021.
  • cells were mechanically dissociated and seeded on dishes pre-coated with polyornithine (15 ⁇ g ml "1 ) and laminin (4 ⁇ g ml “1 ) with a splitting ratio of 1 :2 in N2/B27 supplemented with 20 ng ml "1 BDNF, 20 ng ml "1 GDNF, 0.2 mM ascorbic acid, 1 ng ml "1 ⁇ 3, 0.5 mM dibutyryl cAMP and 10 ⁇ DAPT.
  • Example 7 Immunofluorescence images of neural induction on MS5 cells and iHLCs co-cultured with hiPSCs.
  • hiPSCs were compared to the differentiation of hiPSCs on MS5 cells. Briefly, hiPSCs were obtained and co-cultured on human iHLC in comparison to MS5 cells (as a control) for neural induction. After neural induction, hiPSCs were reseeded on P/0+ LAM/FN for further maturation.
  • Example 8 Immunofluorescence images of neural induction on hepatocyte- derived matrix in comparison to Geltrex-based differentiation.
  • hiPSCs were obtained and plated on hepatocyte-derived matrix (iHLC matrix) and on Geltrex matrix (as a control) for neural induction. After neural induction, hiPSCs were reseeded on P/0+ LAM/FN for further maturation. After 27 days as well as after 50 days of differentiation immunocytochemistry using antibodies against TH and FoxA2 and/or Map2 was applied. A detailed description of hiPSC differentiation on iHLC matrix and on Geltrex matrix is provided in Example 6.
  • Example 9 Expression of markers of early and late neuronal fate acquisition on hepatocyte versus Geltrex matrix.
  • hiPSC were obtained and plated on hepatocyte-derived matrix of human iHLC and on Geltrex matrix (as a control) for neural induction. After neural induction, hiPSCs were reseeded on P/0+ LAM/FN for further maturation. mRNA was then isolated and semi-quantitative PCR of FoxA2, Otx2, Lmxl a, TH, beta3 Tubulin and Map2 as markers of early and late neural fate acquisition was performed at day 7, 13 and 21 of differentiation. A detailed description of the hiPSC differentiation on iHLC matrix and Geltrex matrix is provided in Example 6.
  • Real time PCR shows slightly changes in relative mRNA expression of early neural fate markers such as FoxA2 and Otx2 at day 7 and day 13 for hepatocyte-derived matrix compared to Geltrex matrix.
  • Relative mRNA expression of markers of dopaminergic neurons such as TH and late neuronal fate acquisition such as Map2 shows moderate to strong changes after 21 days of differentiation for hepatocyte-derived matrix compared to Geltrex matrix (Fig. 6).
  • Example 10 No induction into neural fate for human hepatocyte cell line HepG2.
  • the induction of neural fate was performed by testing HepG2 cells as feeder cells in comparison to the differentiation of hiPSCs on MS5 cells.
  • HepG2 were taken as another co-culture system to prove, if other cells besides said iHLCs of the present invention have the potential to lead to the same neural inducing effect.
  • HepG2 cells do not induce neural fate to hiPSCs.
  • hiPSCs were obtained and co-cultured on human HepG2 cells in comparison to MS5 cells for differentiation into neural fate. Images of phase contrast at day 5, 1 1 and 18 were taken. A detailed description of the methods used is provided in Example 4 (instead of hiPSC differentiation on iHLCs, a differentiation of hiPSC on HepG2 cells was performed) and 5. Neuroectoderm rosettes were depicted at day 1 1 and day 18 in MS5 and no neural induction was identified in HepG2 based co-culture system (Fig. 7A).
  • Example 11 Differentiation of hiPSCs on hiPSCs-derived cardiomyocytes, (mesoderm), mesenchymal progenitor cell (mesoderm) or HepG2 cells (endoderm).
  • meoderm mesenchymal progenitor cell
  • HepG2 cells endoderm
  • hiPSCs were plated either on MS5 cells (positive control) or on other cell types such as hiPSCs-derived cardiomyocytes (CMs), mesenchymal progenitor cells (MPCs) or HepG2 cells (HepG2) and observed at days 1 , 4, 5, 7 and 13 post-differentiation. Neuroepithelial rosettes were observed in the MS5 conditions at day 1 1 but not in the other conditions, where only morphological changes were detected.
  • neural induction is not supported by other cell types such as HepG2 cells, CMs and/or MPCs.
  • neural induction is supported in the present invention by hiPSCs-derived iHLCs and iHLC matrix.
  • Example 12 Differentiation of hiPSCs on Geltrex using conditioned medium from HepG2 cells.
  • HepG2 conditioned medium was produced as followed: Coating of dishes for HepG2 conditioned medium with 10 ⁇ g cm 2 Collagen I. Then, HepG2 cells were seeded (50.000 cells per cm 2 ) in 6 ml N2B27 1 :1 medium on a 6 cm dish (1 ,75 x 10 5 cells/ml).
  • N2B27 1 :1 N2 medium: DMEM/F12/, 1x N2, 5 ⁇ g ml insulin, 2 mM L-Glutamine, 1 x non-essential amino acids, 10 ⁇ 2-mercaptoethanol, 50 U/ml Pen, 50 ⁇ g ml Strep; B27 medium: Neurobasal, 1 x B27, 2 mM L-Glutamine, 50 U/ml Pen, 50 ⁇ g/ml Strep) conditioned medium was collected every 3 days from HepG2 cells and iHLCs, mixed 1 :1 with fresh N2B27 1 :1 medium and used for differentiation experiments.
  • hiPSCs were dissociated by accutase and seeded on Geltrex coated dishes in mTeSRI supplemented with 10 ⁇ Rock inhibitor Y-27632. After three days, cells were 60-70% confluent and medium was changed to N2B27 1 :1 medium only, to N2B27 1 :1 medium supplemented with 100 nM LDN and 10 ⁇ SB and to N2B27 1 :1 HepG2 conditioned medium
  • N2B27 1 :1 conditioned medium was supplemented with 100 ng ml "1 SHH C24II (Miltenyi) and 100 ng ml "1 FGF8 (Miltenyi).
  • At day 14 of differentiation cells were dissociated mechanically in clumps and were seeded on Poly-L-ornithine (15 ⁇ / ⁇ ) and laminin (5 ⁇ / ⁇ ) coated dishes in N2B27 1 :1 conditioned medium supplemented with 100 ng ml "1 SHH C24II, 100 ng ml "1 FGF8 and 100 ng ml "1 FGF2 (Fig. 11 ).
  • Example 13 Differentiation of hiPSCs to definitive endoderm.
  • hiPSCs were dissociated by accutase and seeded on geltrex coated dishes in mTeSRI supplemented with 10 ⁇ Rock inhibitor Y-27632. Definitive endoderm differentiation was initiated by changing the medium to RPMI containing 1 % B27 w/o insulin supplemented with 100 ng ml "1 FGF2 (Miltenyi) and 100 ng ml "1 Activin A (Miltenyi).
  • Example 14 Differentiation of hiPSC on hiPSCs-derived definitive endodermal cells.
  • Example 15 Differentiation of hiPSCs to mDA neurons in a matrix based differentiation system.
  • Example 16 Differentiation of hiPSC on iHLC matrix vs. HepG2 matrix with dual SMAD inhibition.
  • Example 17 Differentiation of hiPSC on iHLC matrix vs. HepG2 matrix without dual SMAD inhibition.
  • N2B27 1 :1 medium N2 medium: DMEM/F12/, 1 x N2, 5 ⁇ g ml insulin, 2 mM L-Glutamine, non-essential amino acids, 10 ⁇ 2-mercaptoethanol, 50 U/ml Pen, 50 ⁇ g ml Strep; B27 medium: Neurobasal, 1 x B27, 2 mM L-Glutamine, 50 U/ml Pen, 50 ⁇ g/ml Strep
  • Example 18 Differentiation of hiPSCs-derived neural progenitor cells (NPCs) on P/0+ LAM/FN (gold standard), iHLC or HepG2 matrix.
  • iHLC matrix is able to support neuronal maturation at the later stages, preferably dopaminergic maturation.
  • human neural progenitor cells NPCs
  • iHLC matrix is able to support dopaminergic neuron maturation more efficiently in comparison to HepG2 matrix and the gold standard P/O LAM/FN.
  • Example 19 Proteomics data and their confirmation by PCR.
  • ECM proteins which showed a high peptide number in iHLCs as well as in MS5 in comparison to HepG2 and hiPSCs, were selected as candidate proteins (marked in red, Hspg2, Thbsl , Nid 1 , Col5a1 , Ltbpl , Col5a2, Vcan, Emilin-2, Emilin-1 and Fbln2) (Fig. 29).
  • ECM protein combination which includes at least: Perlecan (Hspg2), Thbsl , Nidi , Ltbpl , Col5a1 , Vcan, Larmbl , Lamb2, Lamb3, Larnd , Lamc2, Lamal and/or Lama5.
  • a matrix supporting neural induction contains Lama5, Larmbl , Lamb2, Larnd , Perlecan, Thbsl , Nidi and Vcan, whereas a matrix supporting dopaminergic neuron induction contains Lamal , Lamb3, Lamc2, Ltbpl and Col5a1.
  • Example 20 Up- and downregulation of core matrisome and matrisome associated proteins in iHLCs in comparison to hiPSCs, HepG2 cells and MSCs.
  • Immature HLCs were analyzed by liquid chromatography (LC) with mass spectrometry (MSymass spectrometry (MS) (LC-MS/MS) (MS: Quexative HF mass spectrometer, Themo Fisher Scientific, Waltham, USA; HPLC: Ultimate 3000 RSLC nano-HPLC, Dionex, Sunnyvale, USA) in comparison to hiPSCs, HepG2 cells and MSCs. Relations of significantly up-regulated proteins are illustrated by a venn-diagram. 18 proteins were significantly higher expressed in iHLCs in comparison to all control groups (Fig. 36).
  • 18 proteins being significantly higher expressed by iHCLs in comparison to the controls also include proteins involved in either TGF3- signalling (FBN2, IGFBP2 and 5, LTBP1 ), BMP-signalling (GDF6) or CNS development (AGRN, HAPLN 1 , LAMB1 ). Furthermore proteins, which were only up-regulated relative to two control cell types, could be also assigned to either TGF3-signalling or CNS development.
  • SFRP1 significantly up-regulated in relative to MSC and HepG2 cells, is involved in dopamine neuron development.
  • iHLCs showed a higher expression of FBLN1 , THBS1 (TGF3- signalling) and COL3A1 , COL4A1 , TNC (CNS development) compared to hiPSC and HepG2.
  • THBS1 TGF3- signalling
  • COL3A1 , COL4A1 , TNC CNS development
  • MDK and VTN were significantly higher expressed in iHLCs in comparison to MSC and hiPSCs and are associated with CNS development (Fig. 37).

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Abstract

La présente invention concerne l'utilisation de cellules de type hépatocytes immatures (iHLC) et/ou d'une matrice extracellulaire (ECM) dérivée desdites iHLC en tant que système de culture in vitro pour la différenciation de cellules souches précurseurs cibles en cellules cibles d'un type cellulaire présélectionné, ainsi qu'une composition comprenant lesdites iHLC et/ou ladite ECM en tant que système de culture. La présente invention concerne également un procédé de préparation d'une ECM sans cellule dérivée d'iHLC ainsi qu'un dispositif comprenant des iHLC et/ou une ECM dérivée desdites iHLC en tant que système de culture. La présente invention comprend également un procédé in vitro permettant d'obtenir des cellules cibles d'un type cellulaire présélectionné.
PCT/EP2018/058812 2017-04-06 2018-04-06 Nouvelle matrice dérivée d'hépatocytes humains pour la différenciation de cellules souches et la réparation de tissus Ceased WO2018185260A1 (fr)

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EP4206320A4 (fr) * 2020-08-21 2025-02-12 JSR Corporation Procédé de culture de cellules souches pluripotentes induites humaines, culture de cellules souches pluripotentes induites humaines et procédé de production d'organoïdes cérébraux

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WO2020117031A3 (fr) * 2019-05-23 2020-07-30 Innopeutics Corporation Composition et méthode d'inhibition de l'accumulation et/ou de l'agrégation de bêta-amyloïdes
US11498957B2 (en) 2019-05-23 2022-11-15 Innopeutics Corporation Composition and method for inhibiting amyloid β accumulation and/or aggregation
EP4206320A4 (fr) * 2020-08-21 2025-02-12 JSR Corporation Procédé de culture de cellules souches pluripotentes induites humaines, culture de cellules souches pluripotentes induites humaines et procédé de production d'organoïdes cérébraux

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