WO2013124309A1

WO2013124309A1 - Direct reprogramming of somatic cells into neural stem cells

Info

Publication number: WO2013124309A1
Application number: PCT/EP2013/053366
Authority: WO
Inventors: Dong Wook Han; Hans R. Schöler; Natalia TAPIA
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2012-02-20
Filing date: 2013-02-20
Publication date: 2013-08-29

Abstract

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 POL) family member, wherein the POU family member is not Oct4; and (b) culturing the cells for at least about 2 days. 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. Furthermore, 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 as compared to healthy subjects.

Description

Direct Reprogramming of Somatic Cells into Neural Stem Cells

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. 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. Furthermore, 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.

In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Cell-based regenerative therapy requires the generation of specific cell types for replacing tissues damaged by injury, disease or age. Embryonic stem cells (ESC) 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. However, 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.

A recent approach to overcome these problems was the generation of induced pluripotent stem cells (iPSC). The over-expression of four transcription factors (Oct4, Sox2, Klf4 and c- Myc) was found to be able to reprogram somatic differentiated cells into pluripotent stem cells that in turn can be differentiated into the required cell type (Wu and Hochedlinger, 2011 ). The iPSC technology allows the generation of patient-specific iPSC, bypassing thus the immune rejection obstacles encountered with ESC and further avoiding the destruction of human embryos. Nevertheless the lack of robust lineage-specific differentiation protocols that generate sufficient quantities of specific cell types remains one of the major barriers prior to the implementation of the iPSC approach for therapeutic purposes. 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). However, it is not known whether cell type- specific transcription factors can force somatic cells to acquire different somatic stem cell identities. Recently, the direct reprogramming of differentiated cells into neurons, cardiomyocytes, blood progenitor cells, hepatocytes, and epiblast stem cells (EpiSCs) has been successfully demonstrated by using either the pluripotential reprogramming factors (Oct4, Sox2, Klf4, plus c-Myc; OSKM) or cell type-specific transcription factors together with specific culture conditions (Han et al., 2011 ; Huang et al., 201 1 ; leda et al., 2010; Sekiya and Suzuki, 201 1 ; Szabo et al., 2010; Vierbuchen et al., 2010). Previous studies have shown that combinations of neural transcription factors and/or microRNAs can directly convert both mouse and human fibroblasts into neuronal cells including dopaminergic and motor neurons (Caiazzo et al., 201 1 ; Marro et al., 201 1 ; Pang et al., 2011 ; Pfisterer et al., 201 1 ; Son et al., 201 1 ; Vierbuchen et al., 2010; Yoo et al., 201 1 ). The induced neuronal cells showed neuronal-like gene expression patterns and also generated action potentials, indicating that, in vitro, they are functionally similar to their wild-type counterparts.

However, 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. Furthermore, the authors introduced these exogenous factors using doxycycline inducible lentiviruses, such that the exogenous factors are expressed in the presence of doxycycline. However, in the absence of doxycycline these neural precursor cells are not stably reprogrammed cells, as they are not independent of this exogenous factor. Accordingly, the cells obtained by Lujan et al. 2012 cannot be considered stable NPC that self-renew in the absence of exogenous factors.

Thus, despite the fact that a lot of effort has been invested into methods to direct the reprogramming of somatic cells into neurons or neuronal precursor cells, there is still a need to provide methods of producing stable, self-renewing neural stem cells that overcome the above described disadvantages and that can differentiate in vivo and in vitro into different types of neurons. Such 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.

Accordingly, 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.

The term "induced neural stem cells (iNSC)", as used herein, 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. However, contrary to neural precursor cells (NPC), which can self-renew only for a limited amount of time, NSC have an unlimited ability to proliferate in vivo and in vitro.

Natural NSC as well as methods of obtaining them are well known in the art and have been described, for example in (Reynolds and Weiss, 1992).

The term "somatic cells", as used herein, 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. Preferably, 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 β-cells, hepatic endoderm cells, melanocytes, cord blood endothelial cells, cord blood stem cells, hepatocytes, amniotic cells or liver progenitor 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. Somatic cells of any animal can be employed in the method of the present invention. For example, 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. Preferably, 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. Anokye-Danso et al., 201 1 ; Kim et al., 2009; Okita et al., 2008; Stadtfeld et al., 2008; Warren et al., 2010 and Zhou et al., 2009. In those cases where the compounds are introduced via coding nucleic acid sequences, these 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. Hence, the coding sequences are preferably introduced into a somatic cell by a method achieving high transfection efficiency. For example, 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. Preferably, 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. Preferably, the retroviral MX vector system is used in the method of the invention (Kitamura et al., (2003), Exp Hematol., 31 ( 11 ): 1007-1014).

The term "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. Preferably, 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.

Preferably, 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. It will be appreciated that the different family members employed in the method of the invention may all be from the same species or may be from different species. For example, 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.

The term "Sox family member", as used herein, 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. Preferably, 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.

As used herein, the term "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. Preferably, 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. As used herein, the term "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.

The term "POU family member", as used herein, 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. Preferably, the family members to be employed in accordance with the present invention are Brn4, Pou3f1 , Pou3f2, Pou3f3, Pou4f1 , Pou4f2 and Pou4f3. More preferably, 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.

Most preferably, the combination of factors is selected from Sox2, Klf4, c-Myc and Bm4/Pou3f4.

It will be appreciated that the different compounds or nucleic acid sequences encoding same may be introduced into the somatic cell simultaneously with each other subsequently to each other.

The method of the invention is carried out under suitable cell culture conditions. General cell culture conditions as well as suitable cell culture media are well known in the art (e.g. Cooper GM (2000). "Tools of Cell Biology", ISBN 0-87893-106-6; K. Turksen, ed., Humana Press, 2004, J. Masters, ed., Oxford University Press, 2000, "Animal cell culture", ISBN-10 0-19-963796-2). Suitable culture conditions and media are for example shown in more detail in the Examples of the invention. It is preferred in any of the cell culture conditions described herein that the medium is exchanged (i.e. refreshed) at appropriate intervals, such as e.g. every four days, more preferably every three days, such as e.g. every two days and most preferably the medium is exchanged for fresh cell culture medium every 24 hours.

Preferably, 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). It will be appreciated that the medium components may be replaced by components fulfilling the same function within the medium, such as for example different antibiotics. Furthermore, suitable variations in the amounts of the components can be determined by the skilled person without further ado. For example, 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%. As another example, 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. For example, 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. Furthermore, 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 term "about [...] days", as used herein, encompasses the explicitly recited amount of days as well as deviations therefrom by several hours and/or minutes. In other words, the time of treatment does not have to be exactly the recited amount of days but may differ by several minutes, such as for example 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes etc.. Further, the time of treatment may differ by several hours from the recited amount of days, such as for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours etc.. As an example, the term "about 2 days" encompasses exactly two days, i.e. 48 hours, but also encompasses for example 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours etc. It will be appreciated that the deviation may also be of e.g. 1 hour and 10 minutes or any other combination of the above specifically recited variations of minutes and hours.

The skilled person is aware that such time spans of treatment are relative amounts of time that do not require a 100% accuracy as long as the time span approximately corresponds to the recited amount. Accordingly, a deviation from the recited value of for example 15%, more preferably of 10%, and most preferably of 5% is encompassed by the term "about".

Preferably, the cells are cultured in step (b) for about two days, more preferably for exactly two days. Subsequently, iNSC can be isolated by picking or splitting and further cultured, as described below.

In accordance with the present invention, it was possible for the first time to directly reprogram somatic cells into neural stem cells. This method provides the advantage that there is no need to first induce a pluripotent state that has to be subsequently differentiated into the required cell type. In contrast, the desired cell type is obtained in one step instead of in two costly and time-consuming steps. Moreover, the induced neural stem cells (iNSC) obtained by the method of the present invention can self-renew. Thus, one successful reprogramming event is enough to generate as many cells as needed since the cells continue to proliferate. This is in stark contrast to, for instance, the generation of neurons from somatic cells, as in that case each neuron needs to be derived from a single reprogramming event, due to the lack of proliferation in neurons. In addition, as is shown in the appended examples, 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. Finally, as has also been shown in the appended examples, 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), closely resemble 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. These findings provide the first demonstration that by direct reprogramming a somatic cell can acquire a neural stem cell identity.

In accordance with the present invention, 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. Although 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. Remarkably, 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.

Although the reprogrammed iNSCs were able to suppress the fibroblast-specific transcription network, they still maintained some epigenetic memory of the initial donor cell. Nevertheless, the remaining somatic signature did not impair the functionality of iNSCs both in vitro and in vivo. These results suggest that the newly established NSC transcriptional network is dominant over the remaining fibroblast program.

In a preferred embodiment of the method of the invention, 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.

The term "Tcf/Lef family member", as used herein, 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. Preferably, 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 Roose and Clevers, 1999.

As used herein, the term "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. Preferably, 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.

As used herein, the term "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. Preferably, 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. The term "ASCa and ASCb family member", as used herein, relates to a family of proteins that act as transcriptional activator or repressor. Family-members include for example Mash1/Ascl1 , Mash2 and Mash3. Preferably, 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.

The term "ZEB family member", as used herein, relates to a family of proteins that act as transcriptional activator or repressor. Family-members include for example Sip1 and ZEB1. Preferably, 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.

All other definitions provided herein with regard to the method of the invention, such as e.g. methods of introducing the compounds or cell culture conditions, apply mutatis mutandis also to this preferred embodiment.

More preferably, 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.

In an even more preferred embodiment of the method of the invention, the at least one factor is a Tcf/Lef family member, most preferably E47/Tcf3.

Most preferably, the combination of factors is Sox2, Klf4, c-Myc, Brn4/Pou3f4 and E47 Tcf3.

In another preferred embodiment of the method of the invention, iNSC obtained in step (b) are further expanded.

The term "expanding", in accordance with the present invention, refers to a multiplication of cells, thus resulting in an increase in the total number of cells. Preferably, 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. For example, 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. Preferably, cells are split between 1 :5 and 1 :10 every five to seven days.

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.

As an alternative exemplary method of isolating iNSC, 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. 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), on 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. Such methods are known to the person skilled in the art and have been described, for example in Dainiak et al., 2007(Adv Biochem Eng Biotechnol. 2007;106:1-18). An exemplary method of isolation of iNSCs using a sorting approach has been detailed in Example 6, below.

Preferably, the cells are expanded in cell culture dishes coated with an agent that enhances attachment of cells to the dish. Such 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. Preferably, the dishes are gelatine- or laminin-coated dishes.

In accordance with the present invention, the iNSC are characterised by a high similarity to natural NSC. Preferably, 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.

In another preferred embodiment of the method of the invention, 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.

These 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.

All of the marker proteins referred to herein are defined in accordance with the pertinent prior art.

"SSEA1 ", as used throughout the present invention, 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. In accordance with the present invention, "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.

"Nestin", as used throughout the present invention, 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.

In accordance with the present invention, "SOX1" and "SOX2" refer to Sex determining region Y-box 1 and Sex determining region Y-box 2, which are proteins that in humans are encoded by the SOX1 and SOX2 genes, respectively. SOX1 and SOX2 are transcriptional regulators. Human SOX1 is represented by the NCBI reference NP_005977.2, and SOX2 is represented by the NCBI reference NP_003097.1. SOX1 and SOX2 have been described in the art, for example in Alcock et al., 2009. Murine Sox1 and Sox2 are represented by the NCBI reference NP_033259.2 and NP_035573.3 and have been described in the art, for example in Wood and Episkopou, 1999.

In accordance with the present invention, "PAX6" refer 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 ", as used throughout the present invention, 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.

In accordance with the present invention, "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.

"Glast", as used throughout the present invention, refers to glutamate aspartate transporter, a protein that in humans is encoded by the SLC1A3 gene. GLAST is a transporter protein that is the inner mitochondrial membrane. Human GLAST is represented by the NCBI reference NP_001 160167.1 , NP_001160168.1 and NP_004163.3 and has been described in the art, for example in Shashidharan et al., 1994. Murine Glast is represented by the NCBI reference NP_683740.1 and has been described in the art, for example in Harada et al., 2010.

"Gbx2", as used throughout the present invention, 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.

In accordance with the present invention, "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 , and 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, and 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.

In accordance with the present invention, "Nkx6.1" refers to NK6 homeobox 1 , a protein that in humans is encoded by the NKX6-1 gene. NKX6-1 is a transcriptional regulator. Human NKX6-1 is represented by the NCBI reference NP_006159.2 and has been described in the art, for example in Brejot et al., 2006. Murine Nkx6.1 is represented by the NCBI reference NP_659204.1 and has been described in the art, for example in Sander et al., 2000. The term "at least three", as used herein, 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. Preferably, one of said at least three markers is Sox2. For example, the at least three markers may comprise a combination of Sox2, Olig2 and AscH ; Sox2, Sox1 and Pax6 or Sox2, Olig2 and SSEA1.

Preferably, the at least three markers are human or murine marker proteins.

In another preferred embodiment of the method of the invention, the iNSC are characterised by a lack of expression or a reduced expression as compared to endogenous NSC of at least one of the markers Foxgl , Emx1 , Otx2, Irx3, Nkx2.2, Pax3 and Pax7 and/or by the lack of methylation of the second intron of nestin.

"Foxgl ", as used throughout the present invention, 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. In accordance with the present invention, "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.

"Otx2", as used throughout the present invention, 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. In accordance with the present invention, "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.

"Nkx2.2", as used throughout the present invention, refers to NK2 homeobox 2, a protein that in humans is encoded by the NKX2.2 gene. NKX2.2 is a transcriptional regulator. Human NKX2.2 is represented by the NCBI reference NP_002500.1 and has been described in the art, for example in Cheng et al., 2003. Murine Nkx2.2 is represented by the NCBI reference NP_001071 100.1 and NP_035049.1 and has been described in the art, for example in Holz et al., 2010.

"Pax3" and "Pax7", as used throughout the present invention, 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.

The term "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.

Preferably, the at least one marker is a human or murine marker protein. Furthermore, in accordance with this embodiment of the invention, the iNSC can be characterised by a lack of methylation of the second intron of nestin. As has been described in the examples below, the second intron of Nestin was found to be completely unmethylated in iNSCs and in control NSCs, while it was highly methylated in the starting somatic cells, i.e. fibroblasts, indicating that iNSCs had been reprogrammed at the epigenetic level by the defined transcription factors. Means and methods to assess the methylation level of nucleic acid sequences, such as e.g. bisulphite sequencing PCR, are well known and have been described for the second intron of Nestin in e.g. Han et al. 2009. The nucleic acid sequence of the second intron of Nestin is represented by SEQ ID NO: 1. Preferably, in accordance with this embodiment of characterising the iNSC by a lack of expression or a reduced expression as compared to endogenous NSC of at least one of the markers Foxgl, Emx1 , Otx2, Irx3, Nkx2.2, Pax3 and Pax7 and/or by the lack of methylation of the second intron of nestin, the characterisation is by the lack of methylation of the second intron of nestin.

In another preferred embodiment of the method of the invention, the somatic cells are fibroblasts. More preferably, the somatic cells are embryonic fibroblasts.

In a preferred embodiment, the method of the present invention further comprises differentiating the iNSC into: (i) astrocytes; (ii) neurons; or (iii) oligodendrocytes.

Methods of differentiating known neural stem cells into the above recited cell types are known in the art and have been described, e.g. in Conti et al. 2005 or in Neural Stem Cells (Methods in Molecular Biology) (Awakening the Word Series), Zigova T., Sanberg, P.R. and Sanchez-Ramos, J.R. (Editors), 2002, Humana Press Inc.. Accordingly, the skilled person is capable of testing and employing these conditions for achieving the desired differentiation also when using the iNSC of the present invention as the starting point. Non-limiting examples of methods of differentiating the iNSC of the present invention are described below.

Accordingly, the method of the present invention may further comprise (i) culturing the iNSC obtained in step (b) in a NSC expansion medium for about 24 hours; (ii) culturing the cells obtained in step (i) in neural differentiation medium comprising (a) growth factor(s) for about 96 to 144 hours; and (iii-a) culturing the cells obtained in (ii) in neural differentiation medium without growth factors for about 336 to 504 hours or (iii-b) culturing the cells obtained in (ii) in DMEM/F12 supplemented with non-human serum, dbcAMP, Shh and FGF-8 for at least about 120 hours, thereby initiating the differentiation of the iNSC into neurons and oligodendrocytes.

The NSC expansion medium may be any medium known in the art suitable for the expansion of NSC. Preferably, the NSC expansion medium is DMEM/F-12 [1 :1] supplemented with B27, BSA, glutamine, penicillin, and streptomycin, EGF and bFGF. More preferably, 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. be obtained from PAA Laboratories and is typically provided of a mixture consisting of 200 mM L-glutamine, 10,000 Units/ml Penicillin and 10mg/ml streptomycin, of which preferably at least 0.5%, more preferably at least 1% are added to the medium. 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.

As a neural differentiation medium without growth factors for use in (iii-a), any known neural differentiation medium may be employed. Preferably, the neural differentiation medium is DMEM/F-12 [1 :1] supplemented with B27, BSA, glutamine, penicillin, streptomycin, and bFGF. The preferred amounts for these supplements are as defined in the preceding paragraph.

Alternatively, 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 μΜ, more preferably between 10 and 500 μΜ, and most preferably the amount is 100 μΜ. dbcAMP may be obtained from Sigma-Alrich. 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.

It will be appreciated that 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. For example, 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. Alternatively, cell densities may be in the range of about 50.000 to about 250.000 cells/cm², preferably about 100.000 to about 200.000 cells/cm² and most preferably about 150 000 cells/cm².

Upon the change in cell culture conditions in step (iii-a) or (iii.b), further differentiation of the cells into different specialised cells, i.e. neurons and oligodendrocytes, is initiated. Where purified neural or oligodendrocytic cultures are to be obtained, 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.

Alternatively, 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.

Preferred amounts of media supplements are as described herein above. Preferably, for the derivation of dopaminergic neurons, 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.

In another preferred embodiment of the method of the invention, the method further comprises dedifferentiating the iNSC into induced pluripotent stem cells (iPSC).

The term "induced pluripotent stem cells (iPSC)", as used herein, is defined in accordance with the pertinent art and relates to cells that exhibit characteristics similar to embryonic stem cells (ESCs). Said characteristics include, for example, unlimited self renewal in vitro, a normal karyotype, a characteristic gene expression pattern including stem cell marker genes like Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) and stem cell-specific antigen 3 and 4 (SSEA3/4), and the capacity to differentiate into specialized cell types (Hanna, J., et al. (2007). Science 318(5858): 1920-3; Meissner, A., et al. (2007). Nat Biotechnol 25(10): 1 177-81 ; Nakagawa, M., et al. (2007). Nat Biotechnol.; Okita, K., et al. (2007). Nature 448(7151 ): 313-7; Takahashi, K., et al. (2007Cell 131 (5): 861-72; Wernig, M., et al. (2007). Nature 448(7151 ): 318-24; Yu, J., et al. (2007). Science 318(5858): 1917-20; Park, I. H., et al. (2008). Nature 451(7175): 141-6). 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.

A novel method of generating iPSC has been described recently in the patent application WO 2009/144008. Briefly, 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). Accordingly, in a preferred embodiment of the method of the present invention, the method further comprises dedifferentiating the iNSC into induced pluripotent stem cells (iPSC) by the method described in WO 2009/144008. In another preferred embodiment of the method of the present invention, the cells obtained are free or substantially free of pathogens.

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.

As described elsewhere herein, these cells are very similar to natural NSC. However, some differences between natural NSC and the iNSC of the present invention exist, such as e.g. small differences in gene expression, or the fact that the epigenetic memory from the initial donor cell type is maintained. Furthermore, it will be appreciated that the iNSC differ genetically where compounds are introduced via coding nucleic acid sequences and where these nucleic acid sequences are incorporated into the genomic DNA of the somatic cell. Further differences have been discussed in the examples section below.

Furthermore, 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. In particular, 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. in accidents), age or disease such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer, Huntington, multiple sclerosis spinal muscular atrophy, peripheral neuropathy, Hirschsprung's Diesease, 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). In accordance with the invention, 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. This evidences the applicability of the iNSC or iPSC in gene therapy and/or cell therapy. 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. Experimental 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. Further, 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. Preferably, 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.

Suitable methods for producing iPSC have been described herein above. 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.

Preferably, 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.

All of the definitions and preferred embodiments provided herein above with regard to the methods and the cells of the invention also apply mutatis mutandis to this embodiment of the invention.

Most preferably, 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. Preferably, 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. More preferably, 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. In addition, the composition may contain instructions for use.

The figures show:

Figure 1. Direct Reprogramming of Fibroblasts into iNSCs

(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. (D) RT-PCR analysis showed that both 4F and 5F iNSCs have a similar gene expression profile to control NSCs. (E) 4F iNSCs have so far been stably maintained for more than 130 passages. See also Figures 5, 6 and Table 1.

Figure 2. Characterisation of iNSCs

(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 log₂ 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. The bar to the right indicates the scattering density; the higher the scattering density, the darker the colour. Gene expression levels are depicted in log₂ scale. The number of differentially expressed genes is indicated under each scatter plot. (E) DNA methylation status on the second intron of Nestin in fibroblasts, control NSCs, and 4F and 5F iNSCs was assessed by bisulphite sequencing PCR. Open and filled circles represent unmethylated and methylated CpGs, respectively. (F) Expression levels of region specific marker genes. All data are normalised to Gapdh expression and calibrated on the control NSCs, whose expression is considered as 1 for all genes. (G) Expression level of Olig2 in control NSCs and both 4F and 5F iNSCs as percentage to Actin expression. See also Figure 7 and Table 2.

Figure 3. In vitro differentiation potentials of iNSCs

(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. Representative voltage-clamp recordings in response to increasing voltage pluses from neurons derived from control NSCs, 5F iNSCs, and 4F iNSCs after 7 to 16 days of differentiation. Insets represent higher magnification of sodium currents. Both single and multiple action potentials were detected in neurons derived from control NSCs, 5F iNSCs, and 4F iNSCs. (D). Characterisation of neurons derived from control NSCs and iNSCs. Representative immunofluorescence images of differentiated control NCS, 4F iNSCs and 5F NSCs after 4 to 21 days of differentiation. All three NSC types behaved similarly concerning their spontaneous differentiation behaviour and differentiated into all major neuronal subtypes, namely GABAergic and glutamatergic neurons, ChAT⁺ cholinergic and TFT dopaminergic neurons (except that 4F iNSCs did not differentiate into Thf neurons). Dotted VGIutl expression in proximity of Tuj1 ⁺ nerve fibres suggested morphological synapse formation. Scale bars, 50 μιη and 20μηη (lowermost panel). ChAT, choline acetyltransferase; TH, tyrosine hydroxylase; Tuj, β-tubulin class III; VGIuTI , vesicular glutamate transporter type . See also Table 3.

Figure 4. iNSCs Are Functionally Similar to Control NSCs

In vivo transplantation of iNSCs. 1.5 x 10⁵ 5F iNSCs labelled with GFP were stereotactically transplanted into the subventricular zone of an adult mouse. After transplantation, the fate of transplantated iNSCs was analysed.

Figure 5. iNSC Characterization.

(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.10⁵ cells were plated onto gelatin-coated dishes and the total cell number of cells was determined every 24 h. (F) 4x10⁶ 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.

Figure 6. Integration of the Retroviral Transgenes in the Established iNSCs

(A) 4F iNSCs showed integration of all retroviral transgenes used except for Oct4. (B) Expression levels of both endogenous and retroviral factors used for direct conversion into iNSCs. All data are normalized to Gapdh expression and calibrated on the control NSCs, whose expression is considered as 1 for all genes.

Figure 7. Reprogrammed iNSC Retain Somatic Memory of the Initial Donor Cells

Scatter plot comparison of the global gene expression profiles of 5F iNSCs versus 4F iNSCs of early and late passages. Black lines indicate boundaries of 2-fold difference in gene expression levels. The bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression levels are depicted in the log₂ scale. The numbers of differentially expressed genes are indicated under each scatter plot. Figure 8. In vivo Transplantation of iNSCs

(A, B) 1.5 x 10⁵ of 5F iNSCs were stereotactically transplanted into the subventricular zone and two weeks after transplantation, proliferative and undifferentiated iNSCs could be detected with ki67 and Nestin staining, respectively (A). Differentiating iNSCs could be also detected by inactivation of Sox2 and activation of Mashl , respectively (B).

Figure 9. In vivo Differentiation of Transplantated iNSC

(A, B, C) In vivo differentiation potentials of transplanted iNSCs were determined by immunostaining of GFP and cell fate specific markers. Differentiating GFP⁺/Tuj1 ⁺ and GFP Dcx⁺ neurons (A), GFP GFAP⁺ and GFP⁺/NG2⁺ astrocytes (B), and GFP70lig2⁺ and GFP7S100 ⁺ oligodendrocytes (C) were observed.

Figure 10. iNSC and iNdiPSC phase-contrast images

iNdiPSC present a typical round domed-shaped mouse ESC morphology that differs from the elongated initial iNSC colonies

Figure 11. iNdiPSC alkaline phosphatase (AP) staining

iNdiPSC are positive for AP staining as other pluripotent cell lines such as ESC and iPSC. Figure 12. iNdiPSC stain positive for NANOG

The pluripotent-specific protein NANOG was detected in the iNdiPSC by immunofluorescence.

Figure 13. iNdiPSC stain positive for SSEA-1

The pluripotent-specific marker SSEA-1 was detected in the iNdiPSC by immunofluorescence. Figure 14. Expression of endogenous pluripotency markers in iNdiPSC

The expression of endogenous pluripotent markers was measured by qRT-PCR. Two mESC lines (KH and OG2 rosa) were used as positive control and the initial iNSC were used as negative control. All data are calibrated to OG2 rosa mESCs, which is considered to be 1. Error bars reflect the standard error mean based on technical replicates.

Figure 15. Bisulfite sequencing of genomic Oct4 promoter region

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.

Figure 16. iNdiPSC PCR-based genotyping

Both the initial iNSC and the reprogrammed iNdiPSC contain the integration of the pMX-Klf4 transgene, as it is also required in the iNSC generation. However, pMX-Oct4 transgene integration is only detected in the iNdiPSC and not in the iNSC.

Figure 17. Transgene silencing

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. In addition, pMX-Oct4 and pMX-Klf4 used for the iPSC generation are also silenced in the iNdiPSC.

Figure 18. Karyotyping of the iNdiPSC

Both iNdiPSC clones present 40 chromosomes. Figure 19. In vitro differentiation of the iNdiPSC into the endodermal lineage

Immunocytochemistry for the endodermal marker SOX17 showed that the iNdiPSC are able to differentiate in vitro into the endodermal lineage. Nuclei were stained with Hoechst (blue).

Figure 20. In vitro differentiation of the iNdiPSC into the mesodermal lineage

Immunocytochemistry for the endodermal marker SMA showed that the iNdiPSC are able to differentiate in vitro into the mesodermal lineage. Nuclei were stained with Hoechst (blue). Figure 21. In vitro differentiation of the iNdiPSC into the neural lineage

Immunocytochemistry for the neural marker B3-TUBULIN showed that the iNdiPSC are able to differentiate in vitro into the neural lineage. Nuclei were stained with Hoechst (blue).

Figure 22. In vivo differentiation of the iNdiPSC into all three germ layers

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). The examples illustrate the invention:

Example 1 : Material and methods

Mice and derivation of fibroblasts

All 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.

Generation of iNSCs

To generate iNSCs, fibroblasts (5 x 10⁴ 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. 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

For general neural differentiation, 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 μg/ml streptomycin (all from PAA Laboratories), 10 ng/ml EGF and 10 ng/ml bFGF (both from Peprotec).

The next day, the medium was replaced by 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. At day 4-6 of differentiation, the medium was changed to a neural differentiation medium without any growth factors for an additional 14 to 21 days. Alternatively, several other combinations of growth factors/cytokines were supplemented to DMEM/F12 medium after day 6 for efficient differentiation: 1 % fetal calf serum (GIBCO), 100μΜ dbcAMP, 400ng/ml Shh, 100 ng/ml FGF-8 (both from R&D Systems, Minneapolis, MN). For the generation of GFAP-expressing astrocytes, 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. Immunocytochemistry

For immunofluorescence analysis, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences), washed 3 times with PBS (PAA Laboratories), and then incubated in PBS containing 0.1 % Triton X-100, 1 % BSA fraction V (both Sigma-Aldrich), and 10% FBS Gold (PAA Laboratories) for 45 min at room temperature. The cells were then incubated with primary antibodies overnight at 4°C. 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 day after incubation with primary antibodies, the cells were washed with PBS containing 0.1 % BSA and further incubated with secondary antibodies for 60 min at room temperature. Nuclei were detected by Hoechst 33342 (Fluka) staining.

Quantification of differentiated cell types

The differentiation of cells was quantified at day 8 of differentiation. For image acquisition, the Arrayscan^® VTI Live (Thermo Scientific, Cellomics) was used. The efficiency of neuron differentiation was quantified with the Cellomics Bioapplication Neurite Profiling. Likewise, astrocyte as well as oligodendrocyte differentiation was quantified with the Cellomics Bioapplication Cell Health Profiling based on GFAP and 04 immunostaining, respectively.

Whole genome expression analysis

Whole genome expression analysis was performed according to the method described in Han et ai. 2010. Briefly, 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. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (lllumina) and accompanying software. Samples were hybridised as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (lllumina); background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model; variance stabilisation was performed using the log₂ scaling; and gene expression normalisation was calculated using the quantile method implemented in the lumi package of R-Bioconductor. Data postprocessing and graphics were performed using in-house developed functions in Matlab. 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.

Accession numbers

Microarray results are accessible at the GEO database, with accession number GSE30500. DNA methylation analysis

To determine the DNA methylation status of iNSCs, 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. Briefly, PCR amplifications were performed using SuperTaq polymerase (Ambion) in a total volume of 25 μΙ 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 μΙ of product from the first round of PCR was used in the second round of PCR. The amplified 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).

Electrophysiology

Cells were investigated 7 to 16 days after differentiation using standard whole-cell patch- clamp technique at room temperature. Recordings were made in the whole-cell voltage- clamp or current-clamp mode and data were recorded using an Axopatch 200B amplifier and lso2 data acquisition software (Axon Instruments, Union City, CA) as described previously (Hermann ef al. 2004; Hermann et al. 2006). Extracellular solution contained (in mmol/r¹): 142 NaCI, 8.1 KCI, 1 CaCI₂, 6 MgCI_2> 10 HEPES, 10 D-Glucose (pH 7.4). Pipette solution contained (in mmol/l^"1): 153 KCI, 1 MgCI₂, 5 EGTA, 10 HEPES. Using these solutions, borosilicate pipettes had resistances of 3-6 ΜΩ. 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 10⁴ cells per microlitre. The transplantation was performed on male wildtype C57/BI6 mice (12 weeks, -25 g). For surgery, 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 5μΙ syringe. Franklin & Paxinos mouse brain atlas was used for defining the stereotactic coordinates for the SVZ in relation to bregma: anteroposterior: 1 ,6 mm, mediolateral: ±0,84 mm, dorsoventral: -2,5 below skull. The animals were immunosupressed with a daily i.p. administration of cyclosporine A (Neoral, Novartis, 10 mg kg/d) starting 24h before grafting until perfusion.

Perfusion, sectioning and immunohistochemistry

Under deep anesthesia via 2,5 % Avertin, animals 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 μιη 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 β- 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.

Example 2: Induction of NSC Fate on Fibroblasts

To directly reprogram mouse embryonic fibroblasts (MEFs) into NSCs, 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). After several trials, some neuron-like cells were obtained, but no NSC-like cells (Table 1 ). As 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. Within 4-6 weeks of infecting fibroblasts with the factors, Sox2, Klf4, c-Myc, Pax6, Olig2, Brn4, plus E47 (SKMPOBE), 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. It was next attempted to scale down the number of factors in the reprogramming cocktail (SKMPOBEMS) by omitting one gene at a time. Several rounds of infection with distinct reprogramming cocktails were performed, and within 4 to 5 weeks of infection with the Sox2, Klf4, c-Myc, Brn4, plus E47 (SKMBE, also referred to herein as 5F) combination, 2 to 5 NSC-like cell clusters were obtained (Figure 1A, Table 1 ) from which stable cell lines were established that exhibited expression of NSC marker proteins, such as SSEA1/Olig2 and Nestin/Sox2, indicating that fibroblasts could be directly reprogrammed into an NSC-like state with defined factors (Figures 1 B and 5B). Thus, these NSC-like cells were termed "induced NSCs," or "iNSCs".

Furthermore, it was possible to further scale down the number of factors to 4 (Sox2, Klf4, c- Myc, Brn4; SKMB; also referred to herein as 4F), although the number of NSC-like cell clusters (1 to 3 clusters) was lower than that obtained with 5F (2 to 5 clusters) (Table 1 ). Again, this 4-factor combination successfully produced a few cell clusters that looked very similar to both wild-type NSCs (Figure 1 C) and 5F iNSCs (SKMBE) (Figure 1A).

In addition, non-infected fibroblasts maintained in NSC medium during the same time period showed no NSC-like cells, indicating that the 4F and 5F iNSCs did not arise from preexisting neuronal progenitors present in the initial fibroblast culture (Figures 1A and 1 C). 4F iNSCs exhibited NSC-specific marker gene expression as determined by both immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) (Figures 1 B, 1 D and 5B). 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. As expected, 4F and 5F iNSC did not generate teratomas after injection into immunosuppressed mice (Figure 5E). Finally, iNSCs showed integration of all transgenes except for Oct4, thus excluding the possibility that iNSC arose from the differentiation of contaminating iPSCs (Figure 6A). Next the expression levels of the retroviral factors used for direct conversion toward iNSCs and of their endogenous counterparts was analyzed in the established 5F and 4F iNSC cell lines. To the inventors' surprise, 4F iNSCs showed complete silencing of all transgenes used. In addition, although transgenic expression of both c-Myc and E47 couldn't be detected in 5F iNSC, transgenic levels of Sox2, Klf4 and Brn4 were present but at lower levels than 4 days post-infection fibroblasts (Figure 6B), thus indicating a silencing trend. Interestingly, the expression levels of the endogenous factors in both 4F and 5F iNSCs were similar to those of control NSCs, indicating that both 4F and 5F iNSCs have properly activated the endogenous NSC transcriptional network (Figure 6B). In summary, with two reprogramming cocktails, SKMBE and SKMB, it was possible to generate stable cell lines that highly resemble NSCs in morphology, marker gene expression, and most importantly, self-renewing capacity.

Example 3: Direct Conversion Toward iNSCs is A Gradual Process

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. Interestingly, 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). However, those fibroblasts markers that were still highly expressed in the early-passage 4F iNSCs were dramatically suppressed after several passages. In addition, we analyzed the dynamics of the direct reprogramming process and found that several genes that were highly expressed in fibroblasts but not in control NSCs were still highly expressed in the early-passage 4F iNSCs (Figure 7A). However, both 4F and 5F iNSCs of a late-passage expressed those genes at levels similar to control NSCs (Figure 7A). On the other hand, genes that were specifically expressed in control NSCs but not in fibroblasts were still very lowly expressed in the early-passage 4F iNSCs but were activated upon serial passaging to levels similar to control NSCs (Figure 7B). These findings demonstrate that reprogramming is a gradual process in which the donor cell type-specific transcriptional program is silenced over a period of time. Furthermore, 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). These data indicate that the epigenetic memory from the initial donor cell type, in this case from connective tissue cells, remains in the reprogrammed iNSCs, thus excluding neural progenitors as the initial cell of the conversion process. An epigenetic signature specific to NSCs has previously been found - one that regulates Nestin gene expression (Han et al., 2009). In this study, the difference in the DNA methylation status on the second intron of Nestin between Λ/esf/n-expressing and non- expressing cells was described. To investigate whether these epigenetic changes occurred during direct reprogramming of fibroblasts into iNSCs, the DNA methylation status of the second intron of Nestin in parental fibroblasts, control NSCs, and both 4F and 5F iNSCs was assessed (Figure 2E). The second intron of Nestin was found to be completely unmethylated in 4F and 5F iNSCs, as in control NSCs. However, it was highly methylated in the starting fibroblasts, as in Nestin non-expressing pluripotent cell types (Han et al., 2009), indicating that iNSCs had also been reprogrammed at the epigenetic level by defined transcription factors.

In order to determine the regional identity of the generated iNSCs, 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). No expression of 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). Validation of the microarray data by qRT-PCR (Figure 2F) confirmed the bias towards posterior regions with more than 400-fold up-regulation of the marker genes Hoxb2 and Hoxb7 (Giampaolo et al., 1989). Along the dorsal-ventral axis, only the ventral hindbrain marker Nkx6.1 could be detected, whereas other ventral markers, such as Irx3 and Nkx2.2 (Briscoe et al., 2000), were not expressed or strongly down-regulated compared to control NSCs. Notably, the very ventral marker Olig2 was expressed at a very high level, comparable to control NSCs (Figure 2G). The dorsal markers Pax3 (Goulding et al., 1991 ) and Pax7 (Kawakami et al., 1997) could not be detected or were down -regulated (Figure 2F). In conclusion, judging by both microarray and qRT-PCR data, we suggest a rather posterior regionalization of our iNSCs, since both 4F and 5F iNSCs showed strong up- regulation of posterior marker genes with parallel down-regulation of anterior marker genes. Moreover, the iNSCs most probably reflect a more ventral position due to the expression of the ventral hindbrain gene Nkx6.1 and high levels of Olig2, which has been shown to be important for the development of neurons in the ventral spinal cord (Poh et al., 2002).

Example 4: iNSCs Are as Functionally Mature as Control NSCs

These characterizations have revealed that iNSCs exhibit a morphology, as well as gene, protein, and epigenetic profiles similar to control NSCs. The functionality of the iNSCs through electrophysiology and in vitro and in vivo differentiation was additionally analysed. After inducing the differentiation of both 4F and 5F iNSCs into neurons for 7 to 16 days (Figure 3A), whole-cell voltage-clamp recordings were performed to measure the voltage- gated sodium/potassium currents and to assess the cells' ability to generate action potentials (Figure 3C) It was found that wild-type NSCs and 4F and 5F iNSCs could be differentiated into neurons that expressed sodium currents and that were able to generate single as well as multiple action potentials (Figure 3C), indicating that iNSCs are as functionally mature as wild-type NSCs. Second, the multipotency of the 4F and 5F iNSCs was assessed via the cells' ability to differentiate into their daughter cell types. Differentiation of both 4F and 5F iNSCs into astrocytes, neurons, and oligodendrocytes was induced, as evidenced by GFAP, Tuj1 and 04 staining, respectively (Figure 3A). Both 4F and 5F iNSC lines were able to form neurons as well as astrocytes with the same efficiency than control NSCs. However, the differentiation into oligodendrocytes was less efficient in case of the iNSC lines (Figure 3B). As expected, most neurons were either GABAergic, glutamatergic or to a lesser extend cholinergic (Figure 3D). Some neurons in differentiated control NSCs and 5F iNSCs, but not 4F iNSCs cultures expressed tyrosine hydroxylase (TH) as a marker of dopaminergic cells. Vesicular glutamate transporter 1 (VGIuTI ), packing glutamate into synaptic vesicles, was expressed by neurons from all three NSC types suggesting the development of synapses (Figure 3D). Therefore, both morphological and immunocytochemistry analyses of the resulted neurons showed no major differences between control NSCs and 4F and 5F iNSCs.

Example 5: In vivo differentiation potential of iNSC

In order to investigate the in vivo differentiation potential of iNSCs, 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 10⁵ 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). As the cells were transplanted into the subventricular zone, one of the stem cell niches of the adult brain, we expected that at least some of the transplanted cells would retain their neural stem cell identity. Indeed it was possible to detect GFP-positive cells that also expressed the progenitor marker Nestin. Moreover, through staining with the cell cycle maker Ki67, it was observed that some cells retained the ability to proliferate (Figures 4C and 8). Nevertheless, cells that were positive for the NSC marker Sox2 were never detected (Figures 4D and 8) indicating that although some of the transplanted cells were able to maintain a neural progenitor identity for some time, none of them remained as NSCs. During adult neurogenesis, NSCs produce committed neural progenitors that eventually become neurons. Those 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). Following the in vivo differentiation process, 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). Furthermore, the grafted cells had also committed to the glial lineage, as evidenced by the presence of GFP⁺/GFAP⁺ and GFP⁺/NG2⁺ cells (Figures 4E and 9B). Finally, transplanted iNSCs also had the ability to differentiate into oligodendrocytes, as evidenced by the presence of GFP70lig2⁺ and GFP7S100p⁺ cells (Figures 4E and 9C). Taking all these results together, it is concluded that 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 A) Production of ecotropic viruses · TIMING 4 d

(1 ) On day 1 , aspirate the medium of one 80-90% confluent 100-mm gelatin-coated cell culture dish with 293T cells at approx. 80 - 90 % confluency and wash with PBS. As 293T cells are easily detached during the washing steps, all the washing procedures have to be performed very gently.

(2) Add 1 ml of pre-warmed 0.05% (wt/vol) trypsin/EDTA and incubated at 37 °C for 30 seconds. (3) Collect the cells with 5 ml of MEF medium and transfer them into a 15-ml conical tube. Centrifuge the cells at 400g for 5 min and aspirate the supernatant.

(4) Resuspended the cell pellet in MEF medium and plate the cells on gelatin-coated 100- mm cell culture dishes by splitting them in a 1 :5 ratio. Prepare one 100-mm cell culture dish per virus (e.g. five dishes in total).

(5) On the second day, Transfer 582 μΙ of DMEM without any FBS and other supplements into 5 different 1.5-ml tubes, with one tube per virus.

(6) Add 18 μΙ of FuGENE 6 transfection reagent into each tube from Step 5, mix by gently tapping the tube, and incubate at room temperature for 5 min.

(7) Add 3 pg of one single retroviral vector and 3 pg of pCL-Eco plasmid into one 1.5 ml tube from Step 6 and repeat this process for each different virus. Mix gently by tapping the tube and incubate at room temperature for 5 min.

(8) Remove the MEF medium covering the 293T cells and add 10 ml of fresh MEF medium. The 293T cells should be at 70-80% confluency at the time of transfection.

(9) Add the mixture from Step 7 drop-wise onto the cell culture dishes from Step 8 and incubate the cells at 37°C, 5% C02 for 48 h.

(10) On Day 4: Collect the viral supernatant containing the viral particles from every 100- mm dish 48 h after transfection using plastic disposable pipettes and filter the supernatant through a 0.45-pm syringe filter. Note that incomplete filtering may lead to contamination of the viral supernatant with 293T cells.

(11 ) Aliquot the filtered supernatant into 1.5-ml tubes and immediately store the aliquots at -80°C. Avoid repeated freeze-thaw cycles, as they may reduce the activity of the ecotropic viruses. At this point, the protocol may be paused if necessary.

B) Retroviral transduction of MEFs · TIMING 2 d

(12) On day 1 , plate 5 x 10⁴ MEFs per well on a gelatin-coated 6-well plate using MEF medium and incubate the plate at 37°C, 5% C02 for 6-10 h.

(13) In a 15-ml conical tube, add 250 μΙ of viral supernatant from each factor, 6 μΙ of protamine sulphate (6 mg/ml), and bring up to 2.5 ml with MEF medium

(Note: Protamine sulfate should be added to the viral mixture, as it enhances binding of the viral particles to the cells. Alternatively, polybrene can be added to the viral mixture at a final concentration of 4 pg/ml, instead of protamine sulfate.)

(14) Incubate the plates at 37°C, 5% C02 for 48 h.

(Note: The titer of the ecotropic viral particles may vary among the retroviral batches, thus transduction efficiency will vary among the different viral preparations. For this reason, 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.)

Inducing direct conversion into iNSCs « TIMING 4-5 weeks

(15) On day 1 , aspirate the medium containing the viral particles 48 h after transduction, wash the MEFs with 3 ml of PBS three times, and add 1.5-2 ml of fresh NSC medium.

(16) On day 3, aspirate the medium and add 1.5-2 ml of NSC medium.

(17) From day 5 on: change the medium as in Step 16, every other day.

(18) Aspirate the medium when the transduced MEFs reach 90-100% confluency and wash each well with 2 ml PBS. Add 0.5 ml 0.05% (wt/vol) trypsin/EDTA and incubate the plate at 37°C, 5% C02 for 1-2 min. To obtain a single-cell suspension, add 0.5 ml MEF medium to each well and dissociate the cells by pipetting up and down. Transfer the cells into a 15-ml conical tube containing 4 ml of MEF medium. Centrifuge the tube at 400g for 5 min and aspirate the supernatant.

(19) Replate the cells on a gelatin-coated 60-mm dish with 2 ml of NSC medium.

(Note: As the direct conversion of MEFs into iNSCs is time consuming and the efficiency of conversion is relatively low, all the transduced MEFs should preferably be maintained until iNSC clusters are observed. Therefore, it is advantageous to passage the transduced MEFs only once and to transfer all the cells from the one well of a 6- well plate to a 60-mm dish.

Moreover, partially reprogrammed 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.

Also, late-passage MEFs (passage 3-4) facilitate the identification and enrichment of iNSCs, as their proliferation rate is slower.)

Establishing stable iNSCs · TIMING 1 week

(20) Once iNSC clusters are observed, wait until they grow further. Change the medium every other day.

(Note: As the number of iNSC clusters emerging at 4-5 weeks post-transduction is relatively low (1-5 clusters per initial transduced well), it is recommended to maintain all the transduced cells after passaging and not to split the iNSC clusters until they are fully grown (4-5 days).

(21 ) Stable and pure iNSCs can be established by passaging the entire dish in the following manner:

(a) Aspirate the medium, add 0.5 ml 0.05% (wt/vol) trypsin/EDTA, and incubate the plate at 37 °C, 5% C02 for 5 min. Add 0.5 ml MEF medium per trypsin/EDTA- treated well and dissociate the cells by pipetting up and down to obtain a single- cell suspension. Transfer the cells into a 15-ml conical tube containing 4 ml of MEF medium. Centrifuge the tube at 400g for 5 min and aspirate the supernatant. Replate the cells on a single gelatin-coated 60-mm dish with 2 ml of NSC medium.

(b) Maintain the cells until iNSCs become dominant. This could take 1 or 2 passages.

(Note: 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.)

(c) Plate 2.5-5 x 10⁴ established iNSCs on a gelatin-coated 4-well dish plate with 1 ml of NSC medium and confirm the expression of NSC markers such as Sox2 and Nestin by immunocytochemistry.

E) Culturing established iNSCs

(22) Aspirate the medium, add 1 ml of 0.05% (wt vol) trypsin/EDTA, and incubate the plate at room temperature for 10-20 sec.

(Note: 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.)

(23) Harvest the cells with 5 ml of MEF medium and transfer them to a 15-ml conical tube.

Centrifuge the tube at 400g for 5 min and aspirate the supernatant. Re-plate the cells on a gelatin-coated 60-mm dish with 2 ml of NSC medium.

(Note: 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.

(Note: In many cases, 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

(25) Clonal iNSCs lines can be established using either option A or option B:

(A) Neurosphere formation

(i) When iNSC clusters from Step 21 are fully grown, aspirate the medium, add 0.5 ml 0.05% (wt/vol) trypsin/EDTA, and incubate the plate at 37°C, 5% C02 for 5 min. Add 0.5 ml MEF medium per trypsin/EDTA-treated well and dissociate the cells by pipetting up and down to obtain a single-cell suspension. Transfer the cells into a 15-ml conical tube containing 4 ml of MEF medium. Centrifuge the tube at 400g for 5 min and aspirate the supernatant.

(ii) Dissociate the pellet into single cells by pipetting up and down with 3 ml NSC medium and plate the cells on a non-coated 35-mm bacteriological petri dish. Neurospheres are normally observed in 2-3 days.

(iii) After 3-4 days, the neurospheres should be large enough to manually pick up.

Using fire-polished Pasteur pipettes, transfer each neurosphere into one well of a 12- well plate, add 1 ml of NSC medium per well, and incubate the plate at 37°C, 5% C02.

(Note: Prepare both gelatin- and laminin/poly-lysine-coated 12-well plates. Sometimes, the iNSC neurospheres attach more efficiently to laminin/poly-lysine- coated than to gelatin-coated dishes. Therefore, transfer single neurospheres into both gelatin- and laminin/poly-lysine-coated plates.)

Sorting single iNSCs into 96-well plates

When iNSC clusters from Step 21 are fully grown, aspirate the medium, add 0.5 ml 0.05% (wt/vol) trypsin/EDTA, and incubate the plate at 37°C, 5% CO2 for 5 min. Add 0.5 ml MEF medium per trypsin/EDTA-treated well and dissociate the cells by pipetting up and down to obtain a single-cell suspension. Transfer the cells into a 15- ml conical tube containing 4 ml of MEF medium. Centrifuge the tube at 400g for 5 min and aspirate the supernatant. Re-suspend the cells with NSC medium.

(ii) Re-plate the cells on the gelatin-coated 60-mm dish with 2 ml of NSC medium. (Note: This step enriches iNSCs in the mixed population, which consists of non- reprogrammed MEFs and converted iNSCs.)

(iii) When the cells reach 70-80% confluency, harvest the cells and re-suspend them with NSC medium.

(iv) Adjust the FACS sorting machine and sort single iNSCs on the laminin/polylysine-coated 96-well plates. Once the sorted iNSCs become confluent, passage them into the gelatin-coated 12-well plate.

(Note: The size of iNSCs is smaller than that of MEFs. Sort the smaller cells, which typically represent iNSCs) G) Freezing iNSCs · TIMING 1 h

(26) Aspirate the medium, add 0.5 ml of 0.05% (wt/vol) trypsin/EDTA, and incubate the plate at room temperature for 10-20 sec.

(27) Add 0.5 ml MEF medium per trypsin/EDTA-treated well and dissociate the cells by pipetting up and down to obtain a single-cell suspension. Transfer the cells into a 15- ml conical tube containing 4 ml of MEF medium. Centrifuge the tube at 400g for 5 min and aspirate the supernatant.

(28) Re-suspend the cells with MEF medium containing 10% (vol/vol) DMSO at a final concentration of 1 x 10⁶ cells per ml and aliquot 1 ml of cells per freezing vial.

(29) Keep the vials in a cell-freezing container overnight at - 80°C and then transfer into a liquid nitrogen tank the next day.

• TIMING

Steps 1-11 , Production of ecotropic viruses: 4 days

Step 12-14, Retroviral transduction of MEFs: 2 days

Steps 15-19, Inducing direct conversion into iNSCs: 4 weeks

Steps 20-24, Establishing stable iNSCs: 1 week

Steps 25, Establishing clonal iNSC lines: 1 week

Steps 26-29, Freezing iNSCs: 1 h H) TROUBLESHOOTING

Table 4 below provides troubleshooting advice. Example 7: Reprogramming to pluripotency through a somatic stem cell intermediate

In a further experiment, mouse induced neural stem cells (iNSCs) were reprogrammed to pluripotency using two factors Klf4 and Oct4. iNSC and iPSC culture

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). Induced pluripotent stem cells (iPSC) were cultured in 2i embryonic stem cell (ESC) medium which contains Knockout DMEM medium (Invitrogen), 20% Serum Replacement (Gibco), 1 % Fetal Bovine Serum (Biowest), 1x penicillin/streptomycin/glutamine (Gibco), 1x non-essential aminoacids (Gibco), 1x β- mercaptoethanol (Gibco), 1000 U ml^"1 LIF (ESGRO), 1 μΜ PD0325901 (Stemgent) and 3 μΜ CHIR99021 (Stemgent). Viral production

Retroviral particles coding for mouse Oct4 or mouse Klf4 were produced after co- trasnsfecting 2 x 10⁶ 293T cells using 3 μg 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 μg of the packaging plasmid pCL-Eco (Addgene 12371 ). Importantly, 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 μηη filter (Millipore). iNSC infection

10⁵ iNSCs were plated on a gelatin-coated well from a 6-well plate using iNSC medium. Next day, 300 μΙ of the pMX-Oct4 virus plus 300 μΙ of the pMX-Klf4 virus plus 1900 μΙ of iNSC medium plus 2.5 μΙ of sulfate protamine (4 mg/ml) were added to the plated cells. iNdiPSC generation

48 hours after infection, the medium containing the viral particles was removed and replaced with 2 ml of 2i ESC medium. The first iNSC-derived iPSC colonies (iNdiPSC) were observed 14 days after infection. The efficiency range was between 0.005-0.008 %. The experiment was performed twice in duplicates. iNdiPSC characterization

Two iNdiPSC colonies, namely iNdiPSC-1 and iNdiPSC-4, were picked and expanded on mouse feeders. Both colonies present a typical round domed-shaped mouse ESC morphology (Figure 10) that differs from the elongated initial iNSC colonies (Figure 10). Next, the molecular properties of the generated hiPSC lines were characterize. To this end, the expression of several pluripotency markers was first assessed by immunochemistry. The generated iNdiPSC stained positive for the pluripotency markers alkaline phosphatase (Figure 1 1 ), SSEA-1 (Figure 12) and NANOG staining (Figure 13). Secondly, 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). As expected, the initial iNSC were only expressing the endogenous Sox2 and cMyc but not the other pluripotent markers. Furthermore, bisulfite sequencing analysis of the Oct4 promoter region showed that it was demethylated in contrast to the initial iNSC (Figure 15). Next, PCR-based genotyping could only detect the pMX-Oct4 transgene in the iNdiPSC lines and not on the initial iNSC (Figure 16). In addition, the retroviral transgenes used for the iNSC and the iNdiPSC generation were both silenced, as revealed by qRT-PCR (Figure 17). Finally, both iNiPSC clones showed a normal karyotyping (Figure 18). In summary, these results demonstrate that iNSC can be reprogrammed to iPSC after the forced expression of Klf4 and Oct4.

Next, the pluripotent potential of the generated iNdiPSC lines was assessed. The differentiation potential of iNdiPSC in vitro was investigated by embryoid body (EB) formation. EBs were generated by the hanging-drop method and cells were induced to differentiate. After 2 weeks of differentiation, specific cell types of all three germ layers were observed - i.e. cells stained positive for the endoderm marker Sox17 (Figure 19), the mesoderm marker o-smooth muscle actin (SMA) (Figure 20), and the ectoderm marker β- Tubulin lllb (TUJ1 ) (Figure 21 ), as detected by immunofluorescence microscopy. The in vivo differentiation potential of 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.

Table 1. Summary of the different combinations of reprogramming factors

The number of NSC-like clusters per infection, the number of initial iNSC lines, which were generated but could not be maintained, and the number of stably established iNSC lines are indicated. For both SKMBE and SKMB, the data from two different fibroblast lines (C3H and OG2 MEFs) are shown. Region Gene vs. NSCs vs. Fibroblasts

Foxgl N.E

Forebrain Emx1 N.D N.D

Otx2 N.E

En1

Midbrain

Pax2 N.E N.E

Egr2 + -

Gbx2 +^* +

Anterior hindbrain

Hoxb2 +

Hoxa2 + +

Hoxb7 + +*

Spinal cord

Hoxa7 + +

Irx3 - *

Nkx6.1 + +

Ventral

Nkx2.2 N.E

Olig2

Pax3 N.E N.E

Dorsal

Pax7 N.E N.E

Table 2. Microarray analysis of regional marker expression in iNSCs

The increased or decreased levels of markers relative to control NSCs or parental fibroblasts are shown. -: downregulated; +: upregulated; *: <2 fold; N.E: not expressed in reference cell type; **: not expressed in iNSCs, but in reference cell type; =: no change in expression level; N.D: no probe on microarray chip.

No. of cells RMP AP AP size AP duration AHP

Cell type

measured (mV) (%Y (mV) (ms) (mV)

Control NSCs 13 -46±6 54 101 ±18.3 10.2±5.2 11.0±3.7

5F iNSCs 13 -61 ±15 42 116±30.1 12.4±2.5 22.0±12.5

4F iNSCs 16 -59±21 33 131 ±23 7.8±4.0 22.2±5.9

Table 3. Passive membrane properties and action potential characteristics of cells after 7-16 days of differentiation

Shown is average ± s.e.m. after 7-16 days of spontaneous in vitro differentiation. Cells were analysed at room temperature using borosilicate pipettes with resistances of 3-6 ΜΩ. AP, action potential; AHP, afterhyperpolarization; RMP, resting membrane potential. ""Percentage of all cells investigated firing action potentials. Step Problem Possible reason Solution

19 iNSC Low titer of retroviral 1. Prepare every new retroviral batch clusters particles together with a GFP control. First, do not transduce MEFs using the control appear. GFP retrovirus to estimate the transduction efficiency. Use only retroviral batches with the GFP control of at least 80% or higher transduction efficiency.

Presence of partially 2. Discard the plate and restart the reprog rammed process from the transduction step, as endoderm-like cells epithelial-like cells render the

identification of the iNSC clusters more difficult.

20 iNSCs are The small number of Wait until the initial iNSC cluster

not initial iNSCs are lost becomes fully grown. It often takes present during the passaging several days to observe proliferating after the process iNSCs after passaging the initial iNSC first split cluster.

21 iNSCs do Parental MEFs and Transfer the whole iNSCs on

not attach iNSCs differ in their laminin/poly-lysine-coated dishes.

preference for the Once iNSCs become dominant, they coating matrix sometimes detach from gelatin-coated dishes and form neurospheres.

In this case, dissociate the floating neurospheres into single cells by vigorous pipetting and plate them on laminin/poly-lysine-coated dishes.

Table 4. Troubleshooting table for Example 6. References

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Claims

1. 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.

2. The method of claim 1 , further comprising 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.

3. The method of claim 2, wherein the at least one factor is a Tcf/Lef family member.

4. The method of any one of claims 1 to 3, wherein iNSC obtained in step (b) are further expanded.

5. The method of any one of claims 1 to 4, wherein 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.

6. The method of any one of claims 1 to 5, wherein the iNSC are characterised by a lack of expression or a reduced expression as compared to endogenous NSC of at least one of the markers Foxgl , Emx1 , Otx2, Irx3, Nkx2.2, Pax3 and Pax7 and/or by the lack of methylation of the second intron of nestin.

7. The method of any one of claims 1 to 6, wherein the somatic cells are fibroblasts.

8. The method of any one of claims 1 to 7, further comprising differentiating the iNSC into:

(i) astrocytes;

(ii) neurons; or

(iii) oligodendrocytes.

9. The method of any one of claims 1 to 7, further comprising dedifferentiating the iNSC into induced pluripotent stem cells (iPSC).

10. The method of any one of claims 1 to 9, wherein the cells obtained are free or substantially free of pathogens.

1 1. Induced neural stem cells obtainable by the method of any one of claims 1 to 7.

12. Induced pluripotent stem cells obtainable by the method of claim 11.

13. The iNSC of claim 11 or the iPSC of claim 12 for use in medicine or medical/pharmaceutical research.

14. The iNSC of claim 11 for use in producing iPSC.