WO2013124309A1 - Reprogrammation directe de cellules somatiques en cellules souches neurales - Google Patents

Reprogrammation directe de cellules somatiques en cellules souches neurales Download PDF

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WO2013124309A1
WO2013124309A1 PCT/EP2013/053366 EP2013053366W WO2013124309A1 WO 2013124309 A1 WO2013124309 A1 WO 2013124309A1 EP 2013053366 W EP2013053366 W EP 2013053366W WO 2013124309 A1 WO2013124309 A1 WO 2013124309A1
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cells
insc
inscs
family member
cell
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Dong Wook Han
Hans R. Schöler
Natalia TAPIA
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc

Definitions

  • the present application relates to a method for producing induced neural stem cells (iNSC), comprising (a) introducing into somatic cells (i) a Sox family member, (ii) a Klf family member; (iii) a Myc family member; and (iv) a POU family member, wherein the POL) family member is not Oct4; and (b) culturing the cells for at least about 2 days.
  • iNSC induced neural stem cells
  • the present invention further relates to induced neural stem cells obtainable by the method of the invention, as well as their use in producing induced pluripotent stem cells.
  • the present invention relates to the induced neural stem cells for use in medicine or medical/pharmaceutical research, in particular for use in the treatment of a disease or disorder associated with a reduced number of neurons, astrocytes or oligodendrocytes as compared to healthy subjects.
  • Embryonic stem cells have the potential to differentiate in every cell type from the (human) body and have therefore been extensively studied as a source for replacement therapy.
  • ESC cannot be derived in a patient-specific fashion since they are established from cultured blastocysts. Therefore, immune rejection and ethical concerns are the main barriers that prevent the transfer of the ESC technology, and in particular of human ESC technology, to clinical applications.
  • the iPSC technology is based on the over-expression of four specific transcription factors that are present in the pluripotent state, suggesting that it should be possible to convert one cell type into another directly if using the right combination of transcription factors.
  • Transcription factors specific to embryonic stem cells (ESCs) have been described to induce pluripotency in somatic cells (Maherali et al., 2007; Okita et al., 2007; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007).
  • ESCs embryonic stem cells
  • EpiSCs epiblast stem cells
  • a disadvantage of generating terminally differentiated cell types, in particular neurons is that most of the differentiated cells do not proliferate. Due to the low reprogramming efficiencies, the induction experiments need to be scaled up in order to generate enough cells for transplantation. A strategy that may circumvent this hurdle could be the direct reprogramming into self-renewing somatic stem cells or tissue progenitors. In this case, the low reprogramming efficiencies do not represent a limitation since the desired cells can be expanded, as they are proliferative. Another advantage is that the somatic stem cells could be transplanted into the host niches where they would proliferate and differentiate following the endogenous stimulus, thus acquiring a fully mature phenotype. In addition, the cells differentiated from the transplanted progenitors will respond to inflammatory signals and will migrate towards damaged tissue, facilitating the proper engraftment for medical purposes.
  • Kim et al. 201 1 described the reprogramming of fibroblasts into neural precursor cells (NPCs). To this end, the authors used a combination of the factors Oct4, Sox2, Klf4 and c- Myc, i.e. the factors previously described by Takahashi and Yamanaka, 2006, to induce iPSC reprogramming. These factors specific to induce pluripotency were combined with an NPC medium instead of ESC medium. Due to the use of the Oct4 in their reprogramming cocktail, it is expected that the reprogramming occurred via an initial reprogramming of the fibroblasts toward an intermediate pluripotent state before the culture conditions then differentiated the cells towards the neural precursor fate. Most importantly, the neural precursor cells could only be expanded for 3 to 5 passages, thus excluding a permanent self-renewing capacity, which is one of the most important features of somatic stem cell— based applications.
  • Lujan et al. 2012 described the conversion of mouse fibroblasts into self-renewing, tri-potent neural precursor cells.
  • the factors employed in this study were (i) Rfx4, ID4, FoxG1, Lhx2 and Sox2, (ii) FoxG1, Sox2 and Brn2, (iii) FoxG1 and Brn2 and (iv) FoxG1 and Sox2.
  • the authors emphasised the pivotal role of the factor FoxG1.
  • the authors introduced these exogenous factors using doxycycline inducible lentiviruses, such that the exogenous factors are expressed in the presence of doxycycline.
  • neural stem cells provide a useful tool not only in regenerative therapy but also for disease modelling and drug discovery. This need is addressed by the provision of the embodiments characterised in the claims.
  • the present invention relates to a method for producing induced neural stem cells (iNSC), comprising (a) introducing into somatic cells (i) a Sox family member, (ii) a Klf family member; (iii) a Myc family member; and (iv) a POU family member, wherein the POU family member is not Oct4; and (b) culturing the cells for at least about 2 days.
  • iNSC induced neural stem cells
  • iNSC induced neural stem cells
  • NSC neural stem cells
  • iNSC neural stem cells
  • iNSC relates to cells derived from somatic cells by inducing a "forced” expression of certain genes, and that exhibit characteristics of neural stem cells (NSC).
  • Induced neural stem cells are similar to natural neural stem cells in many respects including for example a prolonged self renewal in vitro, chromatin methylation patterns, lack of teratoma formation, morphology, nuclei size, potency and differentiability in vitro and in vivo into specialized neuronal cell types (astrocytes, oligodendrocytes and different types of neurons) as well as the expression of certain marker genes and proteins such as for example Nestin, Sox2 and Olig2.
  • NSC neural precursor cells
  • NSC neural precursor cells
  • somatic cells is defined in accordance with the pertinent art and refers to any cell type in the mammalian body apart from germ cells and undifferentiated stem cells.
  • the somatic cells are fibroblasts, such as for example from skin tissue biopsies or keratinocytes, B cells, T cells, myeloid progenitors, hematopoietic stem cells, adipose-derived stem cells, dermal papilla cells, pancreatic ⁇ -cells, hepatic endoderm cells, melanocytes, cord blood endothelial cells, cord blood stem cells, hepatocytes, amniotic cells or liver progenitor cells.
  • fibroblasts such as for example from skin tissue biopsies or keratinocytes, B cells, T cells, myeloid progenitors, hematopoietic stem cells, adipose-derived stem cells, dermal papilla cells, pancreatic ⁇ -cells, hepatic endoderm cells,
  • Somatic cells to be used in the method of the invention can for example be derived from existing cells lines or obtained by various methods including, for example, obtaining tissue samples.
  • Cells obtained from tissues samples may be employed directly or may be used to establish a primary cell line.
  • Methods to obtain samples from various tissues and methods to establish primary cell lines are well-known in the art (see e.g. Jones and Wise (1997) Methods Mol Biol. 75:13-21 ).
  • Suitable somatic cell lines may also be purchased from a number of suppliers such as, for example, the American tissue culture collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ) or PromoCell GmbH, Sickingenstr. 63/65, D-69126 Heidelberg.
  • ATCC American tissue culture collection
  • DSMZ German Collection of Microorganisms and Cell Cultures
  • PromoCell GmbH Sickingenstr. 63/65, D-69126 Heidelberg.
  • Somatic cells of any animal can be employed in the method of the present invention.
  • the somatic cells may be from a vertebrate, preferably a mammal, more preferably any one of cat, dog, horse, cattle, swine, goat, sheep, mouse, rat, monkey, ape and human.
  • the somatic cells are human or murine cells.
  • the term "introducing” as used in accordance with the present invention relates to the process of bringing the recited compounds or a nucleic acid sequences encoding same into the target cell. Methods for introducing compounds such as e.g. recombinant proteins, plasmids, messenger RNAs or miRNA are well known to the skilled person and have been described in the art, see e.g.
  • nucleic acid sequences are preferably incorporated into the genomic DNA of the somatic cell.
  • This process is generally known as stable transfection and methods for stable transfection are well-known to the person skilled in the art and described, e.g., in Bonetta, L. (2005, Nature Methods 2, 875-883). Due to the low rate of reprogramming events taking place in transfected cells it is advantageous to rely on an efficient stable transfection method.
  • the coding sequences are preferably introduced into a somatic cell by a method achieving high transfection efficiency.
  • transfection efficiencies of at least 30 %, such as at least 50 %, or at least 80 % are preferred.
  • Suitable methods include, for example, lipofection, electroporation, nucleofection, magnetofection or viral vector infection.
  • retroviral vectors are used to achieve stable transfection of the somatic cells as said vectors not only mediate efficient entry of the coding sequences into the target cell but also their integration into the genomic DNA of the target cell.
  • Retroviral vectors have been shown to be suitable for the transfection of a wide range of cell types from different animal species, to integrate genetic material carried by the vector into the respective cells, to express the transfected coding sequences at high levels, and, advantageously, retroviral vectors do not spread or produce viral proteins after their use in such transfection methods.
  • Suitable retroviral vector systems are well-known to the person skilled in the art such as, e.g., retroviral vectors with the MoMuLV LTR, the MESV LTR, lentiviral vectors with various internal promoters like the CMV promoter, preferably with enhancer/promoter combinations that show silencing of transgene expression in embryonic/pluripotent cells.
  • Episomal vector systems like adenovirus vectors, other non-integrating vectors, episomally replicating plasmids can also be used.
  • the retroviral MX vector system is used in the method of the invention (Kitamura et al., (2003), Exp Hematol., 31 ( 11 ): 1007-1014).
  • coding sequence relates to a nucleotide sequence that, upon expression, gives rise to the encoded product.
  • the expression of the coding sequence in accordance with the present invention can readily be effected in connection with a suitable promoter.
  • the coding sequence corresponds to the cDNA sequence of a gene that gives rise upon expression to a factor that contributes to the reprogramming of a somatic cell into an induced neural stem cell, wherein the reprogramming factors in accordance with the method of the invention are as recited herein.
  • the compounds recited herein are introduced as nucleic acid sequences encoding these compounds. It will be appreciated that all the compounds to be introduced in accordance with the method of the invention can be encoded on one nucleic acid molecule or on a plurality of nucleic acid molecules.
  • the family members recited herein are preferably mammalian proteins, more preferably they are human or mouse proteins. It is preferred that a protein derived from the same species as the somatic cell to be reprogrammed is employed, i.e. if a human somatic cell is to be reprogrammed into a neural stem cell, then it is preferred that the recited family member proteins are human proteins. Nonetheless, the use of e.g. a reprogramming factor of one particular organism for the reprogramming of cells derived from another organism is also envisaged in accordance with the present invention. For example, a human reprogramming factor may be employed in combination with a cell derived from e.g. mice or rats and wee versa.
  • the different family members employed in the method of the invention may all be from the same species or may be from different species.
  • all the recited family members my be human proteins or all the recited family members may be mouse proteins or, alternatively, some of the family members can be from one species, such as e.g. human while the remaining family members for use in the method of the invention are from a different species, such as e.g. mouse.
  • Sox family member relates to a family of proteins that act as a transcriptional activator or repressor, regulating different aspects during development.
  • Family-members include for example Sox2, Sry, Sox1 , Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox1 1 , Sox12, Sox13, SoxU, Sox15, Sox17, Sox18, Sox21 and Sox30.
  • the family members to be employed in accordance with the present invention are Sox2, Sox1 , Sox3 and Sox15. More preferably, the family member to be employed in accordance with the present invention is Sox2.
  • Human Sox2 is represented by the NCBI reference NM_003106.3 and NP_003097.1 and has been described in the art, for example in Stevanovic ef a/., 1994 while mouse Sox2 is represented by the NCBI reference NM_01 1443.3 and NP_035573.3 and has been described in the art, for example in Pevny and Lovell-Badge, 1997.
  • Klf family member relates to a family of proteins that act as a transcriptional activator or repressor depending on the promoter context and/or cooperation with other transcription factors.
  • Family-members include for example Klf4, Klf 1 , Klf2, Klf3, Klf5, Klf6, Klf7, Klf8, Klf9, Klf 10, Klf 11 , Klf 12, Klf 13, Klf 14, Klf 15, Klf 16 and Klf 17.
  • the family members to be employed in accordance with the present invention are Klf4, Klf2 and Klf5. More preferably, the family member to be employed in accordance with the present invention is Klf4.
  • Human Klf4 is represented by the NCBI reference NM_004235.4 and NP_004226.3 and has been described in the art, for example in McConnell and Yang, 2010 while mouse Klf4 is represented by the NCBI reference NM_010637.3 and NP_034767.2 and has been described in the art, for example in Shields et al., 1996.
  • Myc family member relates to a family of proteins that act as a transcriptional activator or repressor. Family-members include for example c-Myc, N-Myc and L-Myc. Most preferably, the family member to be employed in accordance with the present invention is c-Myc.
  • Human c-Myc is represented by the NCBI reference NM_002467.4 and NPJD02458.2 and has been described in the art, for example in Dalla- Favera et al., 1982 while mouse c-Myc is represented by the NCBI reference NM_10849.4 and NP__034979.3 and has been described in the art, for example in Mainwaring et al., 2010.
  • POU family member relates to several protein families, i.e. the Pou1 , Pou2, Pou3, Pou4 and Pou6 Class POU families of proteins, that act as a transcriptional activator or repressor.
  • Family-members include for example Brn4/Pou3f4, Pou1f1, Pou2f1 , Pou2f2, Pou2f3, Pou3f1 , Pou3f2, Pou3f3, Pou4f1 , Pou4f2, Pou4f3, Pou6f1 and Pou6f2.
  • the family members to be employed in accordance with the present invention are Brn4, Pou3f1 , Pou3f2, Pou3f3, Pou4f1 , Pou4f2 and Pou4f3.
  • the family member to be employed in accordance with the present invention is Brn4.
  • Human Brn4 is represented by the NCBI reference NM_000307.3 and NP_000298.2 and has been described in the art, for example in Douville et al., 994 while mouse Brn4 is represented by the NCBI reference NM_008901.1 and NP_032927.1 and has been described in the art, for example in Hara et al., 1992.
  • the combination of factors is selected from Sox2, Klf4, c-Myc and Bm4/Pou3f4.
  • the cells are cultured in step (b) in standard NSC medium well known in the art Palmer et al., 1999.
  • Standard NSC medium is composed of DMEM/F-12 [1 :1] supplemented with 2% N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 g/ml BSA (Fraction V; Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL; 2 mM glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin).
  • the medium components may be replaced by components fulfilling the same function within the medium, such as for example different antibiotics.
  • suitable variations in the amounts of the components can be determined by the skilled person without further ado.
  • the amount of e.g. N2 supplement may be between 0.5 and 5% and the amount of B27 supplement may be up to 5%.
  • the amount of EGF or bFGF may be between 1 to 100 ng/ml.
  • the term "at least”, as used herein, refers to the specifically recited amount but also to more than the specifically recited amount or number.
  • the term “at least two days” encompasses also at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten days, such as at least eleven, at least 12, at least 13, at least 14, at least 15, at least 20 days and so on.
  • this term also encompasses exactly two, exactly three, exactly four, exactly five, exactly six, exactly seven, exactly eight, exactly nine, exactly ten, exactly eleven, exactly 12, exactly 13, exactly 14, exactly 15, exactly 20 days and so on.
  • the cells are cultured in step (b) for about two days, more preferably for exactly two days.
  • iNSC can be isolated by picking or splitting and further cultured, as described below.
  • iNSC induced neural stem cells
  • the iNSC of the present invention can differentiate in vitro and in vivo into neurons, oligodendrocytes and astrocytes. Therefore, a single reprogramming event generates the source of three different cell types.
  • the iNSC of the present invention can be engrafted into the brain of a subject, where they recapitulate the endogenous behaviour of naturally occurring NSC.
  • the directly reprogrammed NSCs namely induced NSCs (iNSCs)
  • iNSCs induced NSCs
  • brain tissue-derived NSCs with respect to a number of characteristics, including morphology, expression profile, self-renewing capacity, epigenetic status, differentiation potentials, and in vitro and in vivo functionality.
  • the factor Oct4 which is usually employed in reprogramming attempts, was omitted from the reprogramming cocktail, thereby enabling the direct reprogramming of fibroblasts into NSCs, i.e., without the cells first passing through an intermediate cell fate.
  • Brn4 and Oct4 are both members of the POU factor family, Brn4 could not replace Oct4 in iPSC generation in experiments carried out by the present inventors (data not shown). Thus, the possibility that iNSCs were generated via the differentiation of iPSCs can be excluded.
  • the iNSCs generated by the method of the invention have self-renewed for more than 130 passages so far and were nearly identical to control NSCs in morphology, gene expression profiles, epigenetic features, and even in vitro and in vivo functionality.
  • the iNSCs could successfully be engrafted in the stem cell niches of the mouse adult brain where they not only continued to proliferate, but also differentiated into neurons, astrocytes and oligondendrocytes, demonstrating a bona fide multipotency in vivo, thereby encouraging potential therapeutic applications.
  • the method further comprises introducing into the somatic cells in step (a) one or more factors selected from the group consisting of: (i) a Tcf/Lef family member; (ii) a Pax family member; (iii) a Oligo family member; (iv) a ASCa and ASCb family member; and (v) a ZEB family member.
  • Tcf/Lef family member relates to a family of proteins that act as a transcriptional activator or repressor.
  • Family-members include for example E47/Tcf3, Tcf1 , Lef1 and Tcf4.
  • the family member to be employed in accordance with the present invention is E47/Tcf3.
  • Human E47/Tcf3 is represented by the NCBI references NM_001 136139.2, NP_001 12961 1.1 , NM_003200.3 and NP_003191.1 and has been described in the art, for example in Breslin et al., 2003 while mouse E47/Tcf3 is represented by the NCBI references NM_001 164147.1 , NP_001 157619.1 , NM_001 164148.1 , NP_001157620.1 , NM_001164149.1 , NP_00 157621.1 , NM_00 164 50.1 , NP_001 157622.1 , NM_001164151.1 , NP_001 157623.1 , NM_001 164152.1 , NP_001 157624.1 , NM_001 164153.1 , NP_001 157625.1 , NM_01 1548.4 and NP_035678.3 and has been described in the art, for example in
  • Pax family member relates to a family of proteins that act as a transcriptional activator or repressor.
  • Family-members include for example Pax6, Pax1 , Pax2, Pax3, Pax4, Pax5, Pax7, Pax8 and Pax9.
  • the family members to be employed in accordance with the present invention are Pax6, Pax2, Pax3 and Pax5. More preferably, the family member to be employed is Pax6.
  • Human Pax6 is represented by the NCBI references NM_000280.3 and NP_000271.1 and has been described in the art, for example in Zhang et al., 2010 while mouse Pax6 is represented by the NCBI references NM_013627.5 and NP_038655.1 and has been described in the art, for example in Hill and Hanson, 1992.
  • Oligo family member relates to a family of proteins that act as a transcriptional activator or repressor.
  • Family-members include for example Olig2, Oligl and Olig3.
  • the family member to be employed in accordance with the present invention is Olig2.
  • Human Olig2 is represented by the NCBI references NM_005806.3 and NP_005797.1 and has been described in the art, for example in Jakovcevski and Zecevic, 2005 while mouse Olig2 is represented by the NCBI references NM_016967.2 and NP_058663.2 and has been described in the art, for example in Takebayashi et al., 2000.
  • ASCa and ASCb family member relate to a family of proteins that act as transcriptional activator or repressor.
  • Family-members include for example Mash1/Ascl1 , Mash2 and Mash3.
  • the family member to be employed in accordance with the present invention is Mash1/Ascl1.
  • Human Mash1/Ascl1 is represented by the NCBI references NM_004316.3 and NP_004307.2 and has been described in the art, for example in Letinic et al., 2002 while mouse Mash1/Ascl1 is represented by the NCBI references NM_008553.4 and NP_032579.2 and has been described in the art, for example in Bertrand et al., 2002.
  • ZEB family member relates to a family of proteins that act as transcriptional activator or repressor.
  • Family-members include for example Sip1 and ZEB1.
  • the family member to be employed in accordance with the present invention is Sip1.
  • Human Sip1 is represented by the NCBI references NM_003616.2 and NP_003607 and has been described in the art, for example in Vandewalle et al., 2009 while mouse Sip1 is represented by the NCBI references NM_025656.4 and NP_079932.2 and has been described in the art, for example in Seuntjens et al., 2009.
  • this embodiment comprises introducing into the somatic cells in step (a) one or more factors selected from the group consisting of: (i) E47/Tcf3; (ii) Pax6; (iii) Olig2; (iv) Mash1/Ascl1 ; and (v) Sip1.
  • the at least one factor is a Tcf/Lef family member, most preferably E47/Tcf3.
  • the combination of factors is Sox2, Klf4, c-Myc, Brn4/Pou3f4 and E47 Tcf3.
  • expanding refers to a multiplication of cells, thus resulting in an increase in the total number of cells.
  • cells are expanded to at least twice their original number, more preferably to at least 10 times their original number, such as for example at least 100 times, such as at least 1 ,000 times their original number and most preferably to at least 10,000 times, such as at least 100,000 times their original number.
  • the iNSC obtained in step (b) may be further expanded for at least four weeks as shown in the appended examples, in order to achieve such increases in cell numbers.
  • Expansion of the cells may be achieved by known methods, e.g. by culturing the cells under appropriate conditions to high density or confluence and subsequent splitting (or passaging) of the cells, wherein the cells are re-plated at a diluted concentration into an increased number of culture dishes or onto solid supports. With increasing passage number, the amount of cells obtained therefore increases due to cell division.
  • the skilled person is aware of means and methods for splitting cells and can determine the appropriate time point and dilution for splitting cells.
  • cells are split between 1 :5 and 1 :10 every five to seven days.
  • the iNSC Prior to expansion, the iNSC may be mechanically isolated from the initial culture dish and transferred to a new culture vessel, such as for example a different cell culture dish or flask. Induced neural stem cells can be identified by their morphology and by their formation into cell clusters, which can easily be isolated from the remaining cells. For example, neurospheres formed from iNSCs can be separated as detailed in Example 6 below.
  • Mechanical isolation relates to the manual selection and isolation of cells or cell clusters, where necessary under a microscope and may be performed by methods known in the art, such as for example aspiration of the cells into the tip of a pipette or detaching of the cells using a cell scraper or density gradient centrifugation.
  • the cells may be subjected to methods such as e.g. cell sorting approaches including for example magnetic activated cell sorting (MACS) or flow cytometry activated cell sorting (FACS), panning approaches using immobilised antibodies, high-throughput fluorescence microscopy or the use of density gradients.
  • cell sorting approaches including for example magnetic activated cell sorting (MACS) or flow cytometry activated cell sorting (FACS), panning approaches using immobilised antibodies, high-throughput fluorescence microscopy or the use of density gradients.
  • Any surface protein or combinations of surface proteins selectively expressed i.e. not expressed or not expressed to a significant amount on other cell types present in the culture
  • iNSC as described herein below (for example Sox2; or Sox2, Olig2 and AscH ; or Sox2, Sox1 and Pax6 or Sox2, Olig2 and SSEA1 ) may be employed for this isolation.
  • the cells are expanded in cell culture dishes coated with an agent that enhances attachment of cells to the dish.
  • coating agents as well as methods of using them are well known in the art and include, without being limiting gelatine, poly-L-lysin, laminin, poly- L-ornithin, collagen, tenascin, perlecan, phosphocan, brevican, neurocan, thrombospondin, and fibronectin.
  • the dishes are gelatine- or laminin-coated dishes.
  • the iNSC are characterised by a high similarity to natural NSC.
  • the iNSC have a similarity to natural NSC of between 70 and 99%, such as e.g. between 75% and 99%, such as between 80 and 99% and more preferably between 90 and 99%, such as e.g. between 95 and 99% similarity.
  • the degree of similarity can be determined based on any (or all) of the following characteristics: expression profile, epigenetic status, differentiation potential, and in vitro and in vivo functionality. For example, a complete expression profile of the iNSC may be compared to the expression profile of natural occurring NSC and the degree of similarity may be determined.
  • the iNSC are characterised by the expression of at least three markers selected from the group consisting of SSEA1 , Olig2, Nestin, Sox2, Sox1 , Pax6, Mashl , Blbp, Glast, Gbx2, Hoxb2, Hoxa2, Hoxa7, Nkx6.1 and Hoxb7.
  • markers are characteristic markers of the induced neural stem cells, i.e. they are expressed once these cells have formed. While at least four compounds are introduced into the somatic cells in accordance with the method of the invention, it is not necessary to analyse these four markers in order to characterise iNSC.
  • SSEA1 refers to stage-specific embryonic antigen 1 that in humans is encoded by the FUT4 gene.
  • SSEA1 is a carbohydrate that is present on the surface of the cells.
  • Human SSEA1 is represented by the NCBI reference NP_002024.1 and has been described in the art, for example in Yanagisawa et al., 2011.
  • Murine SSEA1 is represented by the NCBI reference NP_034372.1 and has been described in the art, for example in Yagi et al., 2010.
  • Olig2 refers to oligodendrocyte transcription factor 2 that in humans is encoded by the OLIG2 gene.
  • OLIG2 is a transcriptional regulator.
  • Human OLIG2 is represented by the NCBI reference NP_005797.1 and has been described in the art, for example in Takebayashi et al., 2000.
  • Murine Olig2 is represented by the NCBI reference NP_058663.2 and has been described in the art, for example in Setoguchi and Kondo, 2004.
  • Neestin refers to a protein that in humans is encoded by the NES gene.
  • Nestin is an intermediate filament.
  • Human nestin is represented by the NCBI reference NP_006608.1 and has been described in the art, for example in Michalcyzk and Ziman (Histol. Histopathol. (2005) 20:665-671 ).
  • Murine nestin is represented by the NCBI reference NP_057910.3 and has been described in the art, for example in Han et al., 2009.
  • PAX6 refers to Paired box 6, which is a protein that in humans is encoded by the PAX6 gene.
  • PAX6 is a transcriptional regulator.
  • Human PAX6 is represented by the NCBI references NP_000271.1 , NP_001 121084.1 , NP_001595.2. PAX6 has been described in the art, for example in Strachan and Read (Curr. Opin. Genet. Dev. (1994) 4:427-438).
  • Murine Pax6 is represented by the NCBI reference NP_001231 127.1 , NP_001231 129.1 , NP_001231 130.1 , NP_001231 131.1 and NP_038655.1 and has been described in the art, for example in Xu et al., 1999.
  • “Mashl” refers to achaete-scute complex homolog 1 , a protein that in humans is encoded by the ASCL1 gene.
  • ASCL1 is a transcriptional regulator.
  • Human ASCL1 is represented by the NCBI reference NP_004307.2 and has been described in the art, for example in Pang et al., 201 1.
  • Murine Mashl is represented by the NCBI reference NP_032579.2 and has been described in the art, for example in Parras et al., 2007.
  • Blbp refers to brain lipid binding protein, a protein that in humans is encoded by the FABP7 gene.
  • BLBP is a brain fatty acid binding protein.
  • Human BLBP is represented by the NCBI reference NP_001437.1 and has been described in the art, for example in Kipp et al., 201 1.
  • Murine Blbp is represented by the NCBI reference NP_067247.1 and has been described in the art, for example in Feng et al., 1994.
  • Gbx2 refers to gastrulation brain homeobox 2, a protein that in humans is encoded by the GBX2 gene.
  • GBX2 is a transcriptional regulator.
  • Human GBX2 is represented by the NCBI reference NP_001476.2 and has been described in the art, for example in Lin et al., 1996.
  • Murine Gbx2 is represented by the NCBI reference NPJD34392.1 and has been described in the art, for example in Sunmonu et al., 2009.
  • HOXA2 and HOXB2 refer to Homeobox A2 and Homeobox B2, which are proteins that in humans are encoded by the HOXA2 and HOXB2 genes, respectively.
  • HOXA2 and HOXB2 are transcriptional regulators.
  • Human HOXA2 is represented by the NCBI reference NP_006726.1
  • HOXB2 is represented by the NCBI reference NP_002136.1.
  • HOXA2 and HOXB2 have been described in the art, for example in Davenne et al. (Neuron (1999) 22:677-691 ).
  • HOXA7 and HOXB7 refer to Homeobox A7 and Homeobox B7, which are proteins that in humans are encoded by the HOXA7 and HOXB7 genes, respectively.
  • HOXA7 and HOXB7 are transcriptional regulators.
  • Human HOXA7 is represented by the NCBI reference NP_008827.2
  • HOXB7 is represented by the NCBI reference NP_004493.3.
  • HOXA7 and HOXB7 have been described in the art, for example in Knittel et al., 1995 or Vogels et al., 1990.
  • Murine Hoxa7 and Hoxb7 are represented by the NCBI reference NP_034585.1 and NP_03459.2 and have been described in the art, for example in Chen et al., 1998.
  • At least three encompasses also at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten different markers or more, such as at least eleven, at least 12, at least 13, at least 14, or all 15 of the recited markers. It will be appreciated by the skilled person that this term further encompasses exactly three, exactly four, exactly five, exactly six, exactly seven, exactly eight, exactly nine, exactly ten, exactly eleven, exactly 12, exactly 13, exactly 14, or exactly 15 markers from the recited list of markers.
  • one of said at least three markers is Sox2.
  • the at least three markers may comprise a combination of Sox2, Olig2 and AscH ; Sox2, Sox1 and Pax6 or Sox2, Olig2 and SSEA1.
  • the at least three markers are human or murine marker proteins.
  • Foxgl refers to forkhead box G1 , a protein that in humans is encoded by the FOXG1 gene.
  • FOXG1 is a transcriptional regulator.
  • Human FOXG1 is represented by the NCBI reference NP_005240.3 and has been described in the art, for example in Manuel et al., 201 1.
  • Murine Foxgl is represented by the NCBI reference NP_001 153584.1 and NP_032267.1 and has been described in the art, for example in Regad et al., 2007.
  • “Emx1” refers to empty spiracles homeobox 1 , a protein that in humans is encoded by the EMX1 gene.
  • EMX1 is a transcriptional factor.
  • Human EMX1 is represented by the NCBI reference NP_004088.2 and has been described in the art, for example in Cocas et al., 2009.
  • Murine Emx1 is represented by the NCBI reference NP_034261.1 and has been described in the art, for example in Young et al., 2007.
  • Oxy2 refers to orthodenticle homeobox 2, a protein that in humans is encoded by the 07X2 gene.
  • OTX2 is a transcriptional regulator.
  • Human OTX2 is represented by the NCBI reference NP_068374.1 and NP_758840.1 and has been described in the art, for example in Ang et al., 1996.
  • Murine Otx2 is represented by the NCBI reference NP_659090.1 and has been described in the art, for example in Ang et al., 1996.
  • IRX3 refers to Iroquois homeobox 3, a protein that in humans is encoded by the IRX3 gene.
  • IRX3 is a transcriptional regulator.
  • Human IRX3 is represented by the NCBI reference NP_077312.2 and has been described in the art, for example in Briscoe and Ericson (Current Opinion in Neurobiology (2001 ) 1 :43-49).
  • Murine Irx3 is represented by the NCBI reference NP_001240751 and NP_032419.2 and has been described in the art, for example in Chen et al., 201 1.
  • Pax3 and Pax7 refers to paired box 3 and paired box 7, which are proteins that in humans are encoded by the PAX3 and PAX7 genes.
  • PAX3 and PAX7 are transcriptional regulators.
  • Human PAX3 is represented by the NCBI references NP_000429.2, NP_001 120838.1 , NP_039230.1 , NP_852122.1 , NP_852123.1 , NP_852124.1 , NP_852125.1 and NP_852126.1
  • human PAX7 is represented by the NCBI references NP_001 128726.1 , NP_002575.1 and NP_039236.1 and both have been described in the art, for example in Mansouri and Gruss, 1998.
  • Murine Pax3 is represented by the NCBI references NP_001 15299.1 and NP_032807.3
  • Pax7 is represented by the NCBI reference NP_035169.1 and have been described in the art, for example in Mansouri and Gruss, 1998.
  • At least one in accordance with this embodiment, encompasses also at least two, at least three, at least four, at least five, at least six or all seven of the recited markers. It will be appreciated by the skilled person that this term further encompasses exactly three, exactly four, exactly five, exactly six or exactly seven markers from the recited list of markers.
  • the somatic cells are fibroblasts. More preferably, the somatic cells are embryonic fibroblasts.
  • the NSC expansion medium may be any medium known in the art suitable for the expansion of NSC.
  • the NSC expansion medium is DMEM/F-12 [1 :1] supplemented with B27, BSA, glutamine, penicillin, and streptomycin, EGF and bFGF.
  • the B27 is B27 w/o Vitamin A (e.g. from Invitrogen) and the BSA is fraction V (e.g. from Invitrogen).
  • Preferred amounts of B27 are up to 5%, preferably 2%.
  • Preferred amounts of BSA are between 0.001 to 0.01 %, preferably 0.005%.
  • Glutamine, penicillin and streptomycin may e.g.
  • EGF and bFGF may e.g. be obtained from Peprotec and preferred amounts to be employed are between 0.5 and 50 ng/ml, more preferably between 1 and 25 ng/ml, and most preferably the amount is 10 ng/ml, each.
  • the neural differentiation medium is DMEM/F-12 [1 :1] supplemented with B27, BSA, glutamine, penicillin, streptomycin, and bFGF.
  • B27, BSA, glutamine, penicillin, streptomycin, and bFGF are preferred amounts for these supplements.
  • the cells obtained in (ii) may be cultured in DMEM/F12 supplemented with non-human serum, dbcAMP, Shh and FGF-8.
  • Preferred amounts of serum to be employed are between 0.1 % and 10%, more preferably between 0.5 and 2.5%, and most preferably the amount is 1 %.
  • Serum such as for example FCS may be obtained from e.g. GIBCO or PAA.
  • Preferred amounts of dbcAMP to be employed are between 1 and 1000 ⁇ , more preferably between 10 and 500 ⁇ , and most preferably the amount is 100 ⁇ .
  • dbcAMP may be obtained from Sigma-Alrich.
  • Shh Preferred amounts of Shh, to be employed are between 50 and 1000 ng/ml, more preferably 400ng/ml.
  • Shh may be obtained from R&D Systems, Minneapolis, MN.
  • Preferred amounts of FGF-8 to be employed are between 1 ng/ml and 1000 ng/ml, more preferably between 10 ng/ml and 500 ng/ml, and most preferably the amount is 100 ng/ml.
  • FGF-8 may be obtained from Systems, Minneapolis, MN.
  • the iNSC obtained in step (b) of the method of the invention are to be plated at an appropriate density for any of the further cell cultures referred to herein.
  • cells are seeded at a density of at least 10 cells per well of a 48-well plate, such as e.g. at least 100 cell per will, preferably at least 1.000 cells per well, more preferably at least 10.000 cells per well and most preferably the cells are plated at a density of about 100.000 cells per well.
  • cell densities may be in the range of about 50.000 to about 250.000 cells/cm 2 , preferably about 100.000 to about 200.000 cells/cm 2 and most preferably about 150 000 cells/cm 2 .
  • step (iii-a) or (iii.b) further differentiation of the cells into different specialised cells, i.e. neurons and oligodendrocytes, is initiated.
  • the cells can be separated by well known methods, such as e.g. sorting of cells based on magnetic activated cell sorting (MACS) or flow cytometry activated cell sorting (FACS), panning approaches using immobilised antibodies or the use of density gradients, as described herein above.
  • MCS magnetic activated cell sorting
  • FACS flow cytometry activated cell sorting
  • the method of the present invention may further comprise (i) culturing the iNSC obtained in step (b) in DMEM/F-12 comprising B27, BSA, FCS, glutamine, penicillin and streptomycin for about 172 to about 220 hours, preferably for about 192 hours, thereby differentiating the iNSC into astrocytes.
  • iNSC are employed that were obtained by employing the five factors (i) a Sox family member; (ii) a Klf family member; (iii) a Myc family member; and (iv) a POU family member, wherein the POU family member is not Oct4 and (v) a Tcf/Lef family member. More preferably, the iNSC are obtained by employing the factors (i) Sox2; (ii) Klf4; (iii) c-Myc; and (iv) Brn4/Pou3f4 and, optionally, (v) E47/Tcf3.
  • the method further comprises dedifferentiating the iNSC into induced pluripotent stem cells (iPSC).
  • iPSC induced pluripotent stem cells
  • iPSC The state of the art generation of iPSC from fibroblast cultures has been described in Takahashi, Okita, Nakagawa, Yamanaka (2007) Nature Protocols 2(12).
  • the pluripotency of murine iPSC can tested, e.g., by in vitro differentiation into neural, glia and cardiac cells and the production of germline chimaeric mice through blastocyst injection.
  • Human iPSC lines can be analysed through in vitro differentiation into neural, glia and cardiac cells and their in vivo differentiation capacity can be tested by injection into immunodeficient SCID mice and the characterisation of resulting tumours as teratomas.
  • WO 2009/144008 describes the production of induced pluripotent stem cells by a method comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox.
  • Preferred factors according to WO 2009/144008 belonging to the factor families of Myc, Klf and Sox and endogenously expressed by or encoded by the coding sequences to be introduced into the target cell are selected from the group consisting of l-Myc, n-Myc, c-Myc, Klfl , Klf2, Klf4, Klf15, Sox1 , Sox2, Sox3, Sox15 and Sox18. It is further preferred by said method that the target cell does not endogenously express one of the factors encoded by the one or two coding sequences to be introduced into said target cell. Furthermore, it is preferred that the target cell is a neural stem cell (NSC).
  • NSC neural stem cell
  • Pathogens to be avoided are well known to the skilled person and include, without being limiting, viruses such as for example Hepatitis virus A, B, C, Epstein-Barr-Virus or HIV-Virus and bacteria such as for example mycoplasm or chlamydia.
  • the cells are considered to be essentially free of pathogens if less than 0.01 % of cells comprise pathogens, such as e.g. less than 0.005% of cells, preferably less than 0.001% of cells and most preferably less than 0.0001 % of cells.
  • the present invention also relates to induced neural stem cells obtainable by the method of the invention.
  • the present invention relates to induced pluripotent stem cells obtainable by the method of the invention of further dedifferentiating the iNSC into induced pluripotent stem cells.
  • the present invention further relates to the iNSC or the iPSC of the invention for use in medicine or medical/pharmaceutical research.
  • the cells of the invention as well as compositions comprising these cells can be used in a variety of therapeutic as well as experimental scenarios.
  • the iNSC of the invention have been shown to not induce the formation of teratomas (see example 2) and are more differentiated than totipotent stem cells. Accordingly, there is an overall reduced risk of these cells developing into cancerous cells, which renders them particularly beneficial in regenerative medicine, gene therapy, cell therapy or drug screening.
  • Regenerative medicine can be used to potentially cure any disease that results from malfunctioning, damaged or failing tissue by either regenerating the damaged tissues in vivo or by growing the tissues and organs in vitro and subsequently implanting them into the patient.
  • the iNSC of the invention are capable of differentiating into different neurons as well as astrocytes or oligodendrocytes and, as has been shown in example 5 below, can be employed in neurobiological aspects of regenerative medicine and hence drastically reduce the need for ES cells.
  • these cells are suitable for use in the treatment of a disease or disorder associated with a reduced number of neurons as compared to healthy subjects.
  • Non-limiting examples of such diseases include damage of brain tissue due to injury (such as e.g.
  • age or disease such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer, Huntington, multiple sclerosis spinal muscular atrophy, peripheral neuropathy, Hirschsprung's Dease, DiGeorge syndrome, Waardenburg syndrome, Charcot-Marie tooth disease, familial disautonomia, congenital insensitivity to pain with anhidorsis and pediatric cancers, such as neuroblastoma.
  • Gene therapy which is based on introducing therapeutic DNA constructs for correcting a genetic defect into germ line cells by ex vivo or in vivo techniques, is one of the most important applications of gene transfer.
  • Suitable vectors and methods for in vitro or in vivo gene therapy are described in the literature and are known to the person skilled in the art (Davis PB, Cooper MJ., AAPS J. (2007), 19;9(1 ):E11-7; Li S, Ma Z., Curr Gene Ther. (2001 ), 1 (2):201 -26).
  • somatic cells obtained from a patient could, for example, be genetically corrected by methods known in the art and subsequently be reprogrammed into induced neural stem cells having the ability to differentiate into neurons, astrocytes or oligodendrocytes, respectively, or into even less differentiated induced pluripotent stem cells.
  • induced neural stem cells having the ability to differentiate into neurons, astrocytes or oligodendrocytes, respectively, or into even less differentiated induced pluripotent stem cells.
  • the cells of the invention can also be used to identify drug targets and test potential therapeutics hence reducing the need for ES cells and in vivo studies.
  • mice Animal setups and methods to identify and/or assess effects of a potential drug including, for example, target-site and -specificity, toxicity or bioavailability are well-known to the person skilled in the art.
  • the cells of the invention may also be useful in experimental settings - besides therapeutic applications - to study a variety of aspects related to neuronal differentiation.
  • the cells can further be subject to studies relating to, e.g., gene therapy, gene targeting, differentiation studies, tests for safety and efficacy of drugs, transplantation of autologous or allogeneic regenerated tissue or tissue repair.
  • the present invention also relates to the induced neural stem cells of the present invention for use in producing iPSC.
  • the induced neural stem cells of the present invention are for use in producing iPSC in accordance with the method described in WO 2009/144008.
  • the present invention further relates to a composition, such as e.g. a kit, for cellular reprogramming of somatic cells into induced neural stem cells, the composition comprising or consisting of (i) a Sox family member; (ii) a Klf family member; (iii) a Myc family member; and (iv) a POU family member, wherein the POL ) family member is not Oct4 and optionally one or more factors selected from the group consisting of: (i) a Tcf/Lef family member; (ii) a Pax family member; (iii) an Oligo family member; (iv) a ASCa and ASCb family member; and (v) a ZEB family member.
  • a composition such as e.g. a kit, for cellular reprogramming of somatic cells into induced neural stem cells
  • the composition comprising or consisting of (i) a Sox family member; (ii) a Klf family member; (iii
  • the composition comprises or consists of (i) a Sox family member; (ii) a Klf family member; (iii) a Myc family member; and (iv) a POU family member, wherein the POU family member is not Oct4 and, optionally, (v) a Tcf/Lef family member. More preferably, the composition comprises or consists of (i) Sox2; (ii) Klf4; (iii) c-Myc; and (iv) Brn4/Pou3f4 and, optionally, (v) E47/Tcf3.
  • the composition consists of the factors recited in (i) to (iv) or in (i) to (v) either in proteinaceous form or as a nucleic acid sequence encoding these factors.
  • the composition consists of these factors in form of a coding nucleic acid sequence ready for use in the transduction of a somatic cell, preferably a fibroblast.
  • the composition is for use in the method of the invention.
  • the various components of the composition (e.g. a kit) may be packaged in one or more containers such as one or more vials.
  • the vials may, in addition to the components, comprise preservatives or buffers for storage.
  • the composition may contain instructions for use.
  • A Morphology of an early iNSC cluster generated by a combination of 5 factors (SKMBE), as assessed by bright field microscopy. MOCK corresponds to fibroblasts that were not transduced with the reprogramming cocktails but were maintained under NSC culture conditions.
  • B Immunofluorescence microscopy images of control NSCs and iNSCs (4F and 5F), using antibodies against SSEA1 and Olig2.
  • C Morphology of an early iNSC cluster generated by 4 factors (SKMB), as assessed by bright field microscopy. MOCK corresponds to fibroblasts that were not transduced with the reprogramming cocktails but were maintained under NSC culture conditions.
  • A Heat map from microarray data demonstrating global gene expression pattern in fibroblasts, control NSCs, 4F iNSCs of early and late passages, and 5F iNSCs.
  • the colour bar at the top indicates gene expression in log 2 scale. Red and blue colours represent higher and lower gene expression levels, respectively.
  • B Hierarchical clustering of the cell lines based on the gene expression profiles in A.
  • C, D Pair-wise scatter plot analysis of the global gene expression profiles of 4F iNSCs of early and late passages, and 5F iNSCs versus the parental fibroblasts (C) and control NSCs (D). Black lines indicate boundaries of 2-fold difference in gene expression levels.
  • A Differentiation potentials of 4F and 5F iNSCs into neurons, astrocytes, and oligodendrocytes as determined by immunocytochemistry using antibodies against Tuj1 , GFAP, and 04, respectively.
  • B The efficiency of differentiation into neurons, astrocytes and oligodendrocytes from control NSCs and iNSCs (4F and 5F) was quantified and compared via immunostaining with Tuj1 , GFAP and 04, respectively.
  • C Electrophysiological properties of control NSC- and iNSC-derived neurons.
  • A Morphology of early iNSC clusters generated by different combinations of 7 factors (SKMPOBE, SKMPOBM and SKMPOBS), as assessed by brightfield microscopy.
  • B Immunofluorescence microscopy images of 4F iNSCs and 5F iNSCs, using antibodies against Nestin and Sox2.
  • C Generation of 4F and 5F iNSCs from a different fibroblast line, OG2 MEFs.
  • D The sizes of 4F (passage 99) and 5F (passage 92) nuclei iNSCs are slightly larger than those of control NSCs. The size of randomly chosen 25 cells per each cell line was measured.
  • E Proliferation rate of iNSC lines upon direct reprogramming.10 5 cells were plated onto gelatin-coated dishes and the total cell number of cells was determined every 24 h.
  • F 4x10 6 of control NSCs (passage 20), ESCs (passage 16), and both 4F (passage 97) and 5F (passage 89) iNSCs in duplicates into SCID mice. ESCs but not both control NSCs and iNSCs formed teratomas after 4 weeks of injection.
  • iNdiPSC present a typical round domed-shaped mouse ESC morphology that differs from the elongated initial iNSC colonies
  • iNdiPSC are positive for AP staining as other pluripotent cell lines such as ESC and iPSC.
  • the pluripotent-specific protein NANOG was detected in the iNdiPSC by immunofluorescence.
  • the pluripotent-specific marker SSEA-1 was detected in the iNdiPSC by immunofluorescence.
  • Figure 14 Expression of endogenous pluripotency markers in iNdiPSC
  • the Oct4 promoter is unmentylated in iNdiPSC and methylated in the initial iNSC, results that correlate with the mRNA Oct4 expression. Open and closed circles indicate unmethylated and methylated CpGs, respectively.
  • Expression level of the viral transgenes was measured in the iNdiPSC by qRT-PCR using specific primers. iNSC at d3 after infection were used as a positive control and the initial iNSC were used as negative control. All data are calibrated to iNSC harvested 3 days after infection, which is considered to be 1.
  • the exogenous transgenes used for iNSC generation are already silenced in the initial iNSC.
  • pMX-Oct4 and pMX-Klf4 used for the iPSC generation are also silenced in the iNdiPSC.
  • Microsections of hematoxylin and eosin-stained teratoma formed within 6-8 weeks of injecting nude mice with iNdiPSCs that had differentiated into tissues of all three germ layers: endoderm (gut-like epithelium), mesoderm (muscle and cartilage) and ectoderm (keratinocytes and neural rosettes).
  • endoderm gut-like epithelium
  • mesoderm muscle and cartilage
  • ectoderm keratinocytes and neural rosettes
  • Example 1 Material and methods
  • mice used were either bred and housed at the mouse facility of the Max Planck Institute (MPI) or bought from Harlan or Jackson laboratories. Animal handling was in accordance with the MPI animal protection guidelines and the German animal protection laws. Fibroblasts were derived from embryos at embryonic day (E)14.5 after removing the head and all internal organs.
  • MPI Max Planck Institute
  • E embryonic day
  • fibroblasts (5 x 10 4 cells) were infected with pMX retrovirus expressing the transcription factors Sox2, Klf4, c-Myc, Pax6, Olig2, Brn4, E47, Mashl , Sip1 , Ngn2, and Lim3 in different combinations.
  • iNSCs Cells infected with different combinations of factors for 48 h were cultured in standard NSC medium: DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 pg/ml BSA (Fraction V; Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). After the first mature iNSC clusters appeared, a mature iNSC clump was either manually picked, or passaged and seeded as whole dishes of cells onto either gelatin- or laminin-coated dishes and the medium was changed every 24 h. Differentiation of iNSCs
  • the iNSCs were seeded at a density of 100,000 cells per well on poly-D-lysine or Matrigel-coated dishes in NSC expansion medium: DME /F-12 [1 :1] with 2% B27 w/o Vitamin A (Invitrogen), 0.005% BSA fraction V (Invitrogen), 2 mM glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (all from PAA Laboratories), 10 ng/ml EGF and 10 ng/ml bFGF (both from Peprotec).
  • DME /F-12 [1 :1] with 2% B27 w/o Vitamin A (Invitrogen), 0.005% BSA fraction V (Invitrogen), 2 mM glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (all from PAA Laboratories), 10 ng/ml EGF and 10 ng/ml bFGF (both from Peprotec).
  • neural differentiation medium DMEM/F-12 [1 :1] with 2% B27 w/o Vitamin A, 0.005% BSA fraction V, 2 mM glutamine, 100 U/ml penicillin, and 100 pg/rnl streptomycin, and 10 ng/ml bFGF.
  • the medium was changed to a neural differentiation medium without any growth factors for an additional 14 to 21 days.
  • DMEM/F12 medium 1 % fetal calf serum (GIBCO), 100 ⁇ dbcAMP, 400ng/ml Shh, 100 ng/ml FGF-8 (both from R&D Systems, Minneapolis, MN).
  • GIBCO fetal calf serum
  • FGF-8 both from R&D Systems, Minneapolis, MN
  • the NSC expansion medium was replaced by DMEM/F-12 [1 :1] with 2% B27 w/o Vitamin A, 0.005% BSA fraction V 10% FCS Gold (PAA Laboratories), 2 mM glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin and cultured for 8 days with a medium change of every other day.
  • Primary antibodies consisted of anti-Nestin (Millipore, 1 :200), anti-Sox2 (Santa Cruz Biotechnology, 1 :400), mouse anti-Tujl antibody (Covance, 1 :000), rabbit anti-Tujl (Covance, 1 :2000), rabbit anti-GFAP IgG antibody (Dako), mouse anti-04 (R&D Systems, 1 :100), mouse anti-synaptophysin antibody (Sigma, 1 :400), rabbit anti-Glutamate antibody (Sigma, 1 :2000), rabbit anti-GABA antibody (Chemicon, 1 :500), rabbit anti-CHAT antibody (Chemicon, 1 :500), guinea pig anti-VGIutl antibody (Pel-Freez, 1 :2000), and rabbit anti-TH antibody (Pel-Freez, 1 :500).
  • the differentiation of cells was quantified at day 8 of differentiation.
  • the Arrayscan ® VTI Live (Thermo Scientific, Cellomics) was used.
  • the efficiency of neuron differentiation was quantified with the Cellomics Bioapplication Neurite Profiling.
  • astrocyte as well as oligodendrocyte differentiation was quantified with the Cellomics Bioapplication Cell Health Profiling based on GFAP and 04 immunostaining, respectively.
  • RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin- labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (lllumina) for 18 h according to the manufacturer's instructions.
  • Hierarchical clustering of genes was performed using the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method.
  • the microarray samples for Fig. 1A correspond to our previous works deposited in the GEO database, NSCs (accession numbers GSM314045, GSM314046, GSM314047), fibroblasts (GSM284799, GSM284800, GSM284801 ) and ESCs (accession number GSM284805, GSM284806, GSM284807) and were normalized using the RMA algorithm.
  • genomic DNA was treated with sodium bisulphite to convert all unmethylated cytosine residues into uracil residues using EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer's protocol. All genomic regions selected were then amplified according to the method described in Han ef a/. 2009 and Han et al. 2008.
  • PCR amplifications were performed using SuperTaq polymerase (Ambion) in a total volume of 25 ⁇ and a protocol of a total of 40 cycles of denaturation at 94°C for 30 s, annealing at the appropriate temperature for each target region for 30 s, extension at 72°C for 30 s with a 1 st denaturation at 94°C for 5 min, and a final extension at 72°C for 10 min.
  • Primer sequences and annealing temperatures used were as follows: Nestin 5' enhancer 1 st sense 5 -TAAAGAGGTTGTTTGGTTTGGTAGT-3'; Nestin 5' enhancer 1 st antisense 5'-CTATTCCACTCAACCTTCCTAAAA-3' (394 bp, 45°C); Nestin 5' enhancer 2 nd sense 5'-TAGTTTTTAGGGAGGAGATTAGAGG-3'; Nestin 5' enhancer 2 nd antisense 5'-CTCTTACCCCAAACACAACTAAAAC-3' (188 bp, 55°C). For each primer set, 3 ⁇ of product from the first round of PCR was used in the second round of PCR.
  • PCR products were verified by electrophoresis on 1 % agarose gel.
  • PCR products were subcloned using the PCR 2.1-TOPO vector (Invitrogen) according to the manufacturer's protocol.
  • Reconstructed plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN) and individual clones were sequenced (GATC-biotech, Germany). Clones were accepted if there was at least 90% cytosine conversion and all possible clonalities were excluded based on the criteria from BiQ Analyzer software (Max Planck Society, Germany).
  • Pipette solution contained (in mmol/l "1 ): 153 KCI, 1 MgCI 2 , 5 EGTA, 10 HEPES. Using these solutions, borosilicate pipettes had resistances of 3-6 ⁇ . Seal resistances in the whole cell mode were between 0.1 and 1 GO. Data were analyzed using lso2, Prism4, and Microsoft Excel 97. Resting membrane potentials (RMP) were determined immediately after gaining whole-cell access. Action potentials (APs) were elicited by applying increasing depolarizing current pulses (5 pA current steps). The after-hyperpolarization (AHP) amplitude was measured from peak to beginning of plateau reached during the current injection, and AP duration was measured at half amplitude. Transplantation
  • Cells were trypsinized and resuspended in medium at a density of 5 x 10 4 cells per microlitre.
  • the transplantation was performed on male wildtype C57/BI6 mice (12 weeks, -25 g).
  • animals were deeply anesthetized by intraperitoneal (i.p.) injection of 0.017 ml of 2.5% Avertin per gram of body weight and fixed into a stereotatic frame.
  • Three microlitres of the cell suspension were injected into the subventricular zone (SVZ) over 5 minutes using a Hamilton 7005KH 5 ⁇ syringe.
  • SVZ subventricular zone
  • mice were intracardially perfused with 50 ml 1xPBS following 50 ml 4 % PFA /1 PBS solution.
  • the brains were isolated and postfixed in 4 % PFA /1 PBS solution over night at 4 °C.
  • 40 ⁇ sagittal sections were performed using a Vibratom (Leica VT 1200 S). Free floating sections were permeabilized in TBS 0.1 M Tris, 150mM NaCI, pH 7.4 / 0.5% Triton-X 100 / 0.1 % Na-Azide / 0.1 % Na-Citrate / 5% normal goat serum (TBS+/+/+) for at least 1 h.
  • Sections were incubated with primary antibodies, diluted in TBS+/+/+, for 48 h on a shaker at 4 °C. Following antibodies were used: Tuj1 (1 :600, Covance), GFAP (1 :1000, Sigma-Aldrich), Olig2 (1 :400, Millipore) and S-100 ⁇ - subunit (1 :1000 Sigma-Aldrich). Incubation with TBS+/+/+ containing Alexa-fluorophore conjugated secondary antibodies (Invitrogen) and Hoechst 33342 (Invitrogen) was performed for 2 h at room temperature. Sections were analyzed with a Zeiss LSM 710 confocal microscope.
  • mice embryonic fibroblasts MEFs
  • three stem cell factors were used (Sox2, Klf4, and c-Myc) together with 8 neural-specific transcription factors (Pax6, Olig2, Brn4/Pou3f4, E47ITcf3, Mash1/Ascl1, Sip1, Ngn2/Neurog2, plus Lim3/Lhx3; SKMPOBEMSNL).
  • Pax6, Olig2, Brn4/Pou3f4, E47ITcf3, Mash1/Ascl1, Sip1, Ngn2/Neurog2, plus Lim3/Lhx3; SKMPOBEMSNL After several trials, some neuron-like cells were obtained, but no NSC-like cells (Table 1 ).
  • Ngn2/Neurog2 and Lim3/Lhx3 are transcription factors that are specific for more differentiated cell types, such as motor neurons (Marro et al., 2011 ), it was speculated that they were directing the reprogramming process toward specific differentiated neuronal cell types. For this reason, Ngn2/Neurog2 and Lim3/Lhx3 were excluded from the reprogramming cocktail, and 6 neural factors (Pax6, Olig2, Brn4/Pou3f4, E47/Tcf3, Mash1IAscl1, plus Sip1) were used together with Sox2, Klf4, and c- Myc.
  • NSC-like clusters were obtained ( Figure 5A, Table 1 ). NSC-like cells were also observed when different combinations of factors were used, such as Sox2, Kif4, c-Myc, Pax6, Olig2, Brn4, and with either Mashl (SKMPOBM) or Sip1 (SKMPOBS) ( Figure 5A, Table 1 ). NSC-like cells could be successfully generated from these three combinations (SKMPOBE, SKMPOBM, SKMPOBS) however, after five or six passages they differentiated into neuron-like cells.
  • iNSCs which present a nuclei slightly larger than control NSCs (Figure 5C), could stably be maintained for more than 130 passages in culture with proliferation rates slightly higher but still comparable to that of wild-type NSCs ( Figures 1 E and 5D), demonstrating that the iNSCs had acquired the ability to self-renew.
  • 4F and 5F iNSC did not generate teratomas after injection into immunosuppressed mice ( Figure 5E).
  • iNSCs showed integration of all transgenes except for Oct4, thus excluding the possibility that iNSC arose from the differentiation of contaminating iPSCs ( Figure 6A).
  • the whole genome profile from 5F iNSCs and from an early- and a late-passage 4F iNSCs was analyzed in order to evaluate the reprogramming level of the entire transcriptome.
  • Fibroblasts and wild-type NSCs were used as a negative and positive control, respectively.
  • the global genome heat map indicates a genome-wide conversion from a fibroblast to a NSC transcriptional program ( Figure 2A). Accordingly, hierarchical clustering analysis grouped the 4F and 5F iNSCs closely to the wild-type NSCs, and not to the parental fibroblasts ( Figure 2B).
  • Pair-wise scatter plots showed that the number of differentially expressed genes was lower when the iNSCs were compared to the control NSCs than when they were compared to the initial fibroblasts ( Figures 2C and 2D).
  • NSC markers such as Olig2, Sox2, and Mash1/Ascl1, which were not initially expressed in fibroblasts, showed a similar expression level in iNSCs and control NSCs. Of these genes, Olig2 and Mash1/Ascl1 were not provided exogenously, indicating an activation of the endogenous NSC program.
  • Ctgf (Ivkovic et al., 2003), a marker of connective tissue, and Acta2 (Schildmeyer et al., 2000), a marker of skeletal muscle, were highly expressed in embryonic fibroblasts and not expressed in control NSCs.
  • Cfg and Acta2 were still highly expressed in 4F iNSCs of an early passage, whereas typical NSC markers, such as Sox2, Olig2, and Mash1/Ascl1, were expressed at levels comparable to those in control NSCs ( Figures 2C and 2D).
  • those fibroblasts markers that were still highly expressed in the early-passage 4F iNSCs were dramatically suppressed after several passages.
  • 5F iNSCs which were rather similar to late-passage 4F iNSCs than to early-passage 4F iNSCs, still showed relatively high expression levels of some fibroblast markers, such as Acta2 and Ctgf ( Figures 2C, 2D, and 7C).
  • Some fibroblast markers such as Acta2 and Ctgf ( Figures 2C, 2D, and 7C).
  • the gene expression level of several markers along the anterior-posterior and dorsal-ventral axis of the brain was analyzed.
  • Microarray analysis revealed a strong bias towards expression of anterior hindbrain markers (Gbx2, Hoxb2, Hoxa2) and even more posterior markers (Hoxa7, HoxbT).
  • anterior markers such as Foxgl, Emx1 or Otx2 (Dou et al., 1999; Simeone et al., 1992a; Simeone et al., 1992b) could be detected and midbrain markers, such as En1 (Davis and Joyner, 1988), were down-regulated or only weakly expressed (Table 2).
  • VKIuTI Vesicular glutamate transporter 1
  • iNSCs were transplanted into the subventricular zone of adult mice. Before transplantation, cells were labeled through viral transduction with retrovirus coding for green fluorescent protein (GFP). Approximately, 1.5 x 10 5 5F iNSCs were stereotactically transplanted into the subventricular zone. Two weeks after transplantation, the fate of the transplanted cells was analyzed in fixed sections ( Figure 4A). The grafts typically consisted of a densely packed core, a less densely organized edge of cells and usually a certain fraction of migrating cells that integrate into the rostral migratory stream (RMS) ( Figure 4B).
  • RMS rostral migratory stream
  • progenitor cells express the marker Mashl.
  • Some of the transplanted iNSCs were found to be positive for nuclear Mashl ( Figures 4C and 8), indicating that iNSCs also followed the same sequence of differentiation events as endogenous NSCs.
  • Those GFP + /Mash1 + cells were mainly localized at the edges of the graft. Originating from those edges some grafted cells migrated towards the RMS and integrated into it ( Figure 4B).
  • the migrating cells were positive for the neuronal markers Dcx and TuJ1 , indicating that the grafted cells had committed to the neuronal lineage in vivo ( Figures 4E and 9A).
  • iNSCs have the potential to undergo differentiation both in vitro and in vivo into all neural cell lineages.
  • Example 6 Exemplary step-by-step protocol of the establishment of clonal iNSC lines and troubleshooting guidelines
  • Protamine sulfate should be added to the viral mixture, as it enhances binding of the viral particles to the cells.
  • polybrene can be added to the viral mixture at a final concentration of 4 pg/ml, instead of protamine sulfate.
  • GFP retroviral particles should preferably be prepared in parallel with the other four viruses. For every new viral batch, it is recommended to transduce one well using the GFP viral particles to estimate the viral titer. It is further recommended that only viral batches with a GFP control of at least 80% transduction efficiency are used to directly reprogram MEFs into iNSCs.
  • epithelial-like cells can be present in transduced dishes due to the overexpression of c-Myc and Klf4. As epithelial cells proliferate fast, and thereby render the identification of iNSC clusters more difficult, it might be more straight-forward and less time-consuming to discard the plate in the presence of epithelial cells and restart the process from Step 12.
  • late-passage MEFs facilitate the identification and enrichment of iNSCs, as their proliferation rate is slower.
  • Stable and pure iNSCs can be established by passaging the entire dish in the following manner:
  • iNSCs grow faster than non-reprogrammed MEFs. Thus stable iNSCs can be established after enriching for the iNSCs present in the dish after continuous passaging. Like brain tissue-derived control NSCs, iNSCs easily detach from the gelatin-coated dishes. Therefore, it is recommended to not wash the cells or to wash very gently with PBS to avoid losing any iNSCs into the medium.
  • iNSCs Like brain tissue-derived control NSCs, iNSCs easily detach from the gelatin- coated dishes. Therefore, it is recommended to not wash the cells or to wash very gently with PBS to avoid losing any iNSCs into the medium.
  • iNSCs immediately detach from the plates upon trypsin/EDTA treatment. Therefore, it is recommended to not treat the cells with trypsin/EDTA for more than 30 sec and to add MEF medium without aspirating the trypsin/EDTA. Adding 5 ml of MEF medium is enough for trypsin/EDTA inactivation.) (24) Change the medium every other day until the iNSCs reach 70-80% confluency. iNSCs can be maintained like the control NSCs derived from brain tissue.
  • iNSCs start to form neurospheres and suddenly float when they become dominant during the purification step (refer to Step 21 ). In this case, immediately transfer the floating neurospheres on laminin/poly-lysine - coated plates after complete dissociation into single cells. iNSCs cultured on both gelatin- and laminin/poly-lysine - coated plates exhibit identical characteristics such as global gene expression pattern, epigenetic features, and differentiation potential.) F) Establishing clonal iNSC lines ⁇ TIMING 1 week
  • iNSCs are smaller than that of MEFs. Sort the smaller cells, which typically represent iNSCs. G) Freezing iNSCs ⁇ TIMING 1 h
  • iNSCs Like brain tissue-derived control NSCs, iNSCs easily detach from the gelatin- coated dishes. Therefore, it is recommended to not wash the cells or to wash very gently with PBS to avoid losing any iNSCs into the medium.
  • Steps 1-11 Production of ecotropic viruses: 4 days
  • Step 12-14 Retroviral transduction of MEFs: 2 days
  • iNSC were maintained in DMEM/F-12 (Invitrogen) supplemented with N2 or B27 (Gibco), 10 ng/ml EGF, 10 ng/ml bFGF (both Invitrogen), 50 pg/ml BSA (Fraction V; Gibco), and 1x penicillin/streptomycin/glutamine (Gibco).
  • Retroviral particles coding for mouse Oct4 or mouse Klf4 were produced after co- trasnsfecting 2 x 10 6 293T cells using 3 ⁇ g of pMX-Oct4 (Addgene 13366) together with 3 g of the packaging plasmid pCL-Eco (Addgene 12371 ) or 3 pg of pMX-Klf4 (Addgene 13370) together with 3 ⁇ g of the packaging plasmid pCL-Eco (Addgene 12371 ).
  • 293T cells were cultured using iNSC medium. 48 hours after transfection, the supernatants containing the viral particles were collected and filtered through a 0.45 ⁇ filter (Millipore).
  • the mRNA levels of the endogenous pluripotency markers Klf4, Oct4, Fgf4, Nanog and Rex1 were comparable to those of two different mESC lines as determined by quantitative real time PCR (qRT-PCR) ( Figure 14).
  • the initial iNSC were only expressing the endogenous Sox2 and cMyc but not the other pluripotent markers.
  • bisulfite sequencing analysis of the Oct4 promoter region showed that it was demethylated in contrast to the initial iNSC ( Figure 15).
  • PCR-based genotyping could only detect the pMX-Oct4 transgene in the iNdiPSC lines and not on the initial iNSC ( Figure 16).
  • iPSCs generated from iNSCs was additionally evaluated by teratoma formation. After 6 to 8 weeks of subcutaneous injection of iNdiPSC lines into nude athymic mice, teratomas containing tissues of all three germ layers had formed from all lines analyzed ( Figure 22). Taken together, these data indicate that iPSC lines can be generated from iNSCs and that these iNdiPSC lines exhibit the pluripotent capacity to differentiate into cells of all three germ layers.
  • iNSCs Once iNSCs become dominant, they coating matrix sometimes detach from gelatin-coated dishes and form neurospheres.
  • a homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube.
  • Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/lrx3 cross- repressive loop. Neuron 69, 721-735.
  • Emx1 -lineage progenitors differentially contribute to neural diversity in the striatum and amygdala. J Neurosci 29, 15933-
  • Pluripotential reprogramming of the somatic genome in hybrid cells occurs with the first cell cycle.
  • Olig transcription factors are expressed in oligodendrocyte and neuronal cells in human fetal CNS. J Neurosci 25, 10064- 10073.
  • PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech De 66, 119-130.
  • a conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development. Development 121 , 1077-1088.
  • Pax3 and Pax7 are expressed in commissural neurons and restrict ventral neuronal identity in the spinal cord.
  • the transcription factor Foxgl regulates telencephalic progenitor proliferation cell autonomously, in part by controlling Pax6 expression levels.
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  • TCF transcription factors molecular switches in carcinogenesis. Biochim Biophys Acta 1424, M23-37.
  • Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev 14, 2134- 2139.
  • Neuron-specific human glutamate transporter molecular cloning, characterization and expression in human brain. Brain Res 662, 245-250.
  • Lysosome-associated membrane protein 1 is a major SSEA-1 -carrier protein in mouse neural stem cells. Glycobiology 20, 976-981.
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Abstract

La présente invention concerne un procédé pour la production de cellules souches neurales induites (iNSC), comprenant (a) l'introduction dans des cellules somatiques (i) d'un membre de la famille Sox, (ii) d'un membre de la famille Klf, (iii) d'un membre de la famille Myc, et (iv) d'un membre de la famille POU, le membre de la famille POU n'étant pas Oct4; et (b) la mise en culture des cellules pendant au moins environ 2 jours. La présente invention concerne en plus les cellules souches neurales induites qu'on peut obtenir par le procédé de l'invention, ainsi que leur utilisation dans la production de cellules souches pluripotentes induites. De plus, la présente invention concerne les cellules souches neurales induites pour l'utilisation en médecine ou dans la recherche médicale/pharmaceutique, en particulier pour l'utilisation dans le traitement d'une maladie ou d'un trouble associé à un nombre réduit de neurones par comparaison aux sujets sains.
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