WO2009093022A2

WO2009093022A2 - Cell re-programming

Info

Publication number: WO2009093022A2
Application number: PCT/GB2009/000164
Authority: WO
Inventors: Jie Na; Peter Andrews
Original assignee: University Of Sheffield
Priority date: 2008-01-23
Filing date: 2009-01-22
Publication date: 2009-07-30
Also published as: WO2009093022A3; GB0801215D0

Abstract

We disclose methods to re-programme differentiated somatic cells to a stem cell phenotype and also the de-differentiation of cancer cells to a cancer stem cell phenotype.

Description

Cell Re-Programming

The invention relates to methods to re-programme differentiated somatic cells to a stem cell phenotype and also the de-differentiation of cancer cells to a cancer stem cell phenotype.

The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be pluripotent or multipotent. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought. A totipotent cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism and are not as such considered stem cells.

Embryonic stem cells (i.e. those having the characteristic of pluripotentiality) may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed embryonic stem (ES) cells. In the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These would ultimately differentiate into germ cells and are referred to as embryonic germ cells (EG cells). Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types but possible differences in behaviour (e.g. with respect to imprinting) have led these cells to be distinguished from one another. Cell culture conditions have been determined which allow the establishment of human ES/EG cells in culture and is described in WO96/22362. A consequence of isolating human ES cells is the destruction of a human embryo. Evidence suggests that tumours are clonal and are therefore derived from a single cell. However, there are few studies that identify and characterize those cells types that are responsible for maintaining tumour cell growth. Some have searched for these so called "cancer stem cells". The concept of a cancer stem cell within a more differentiated tumour mass, as an aberrant form of normal differentiation, is now gaining acceptance over the current model of oncogenesis in which all tumour cells are equivalent both in growth and tumour-initiating capacity [Hamburger AW₁ Salmon SE: Primary bioassay of human tumor stem cells. Science 1977, 197: 461463; Pardal R, Clarke MF, Morrison SJ: Applying the principles of stem cell biology to cancer. Nat. Rev. Cancer 2003, 3: 895902.] For example, in leukaemia, the ability to initiate new tumour growth resides in a rare phenotypically distinct subset of tumour cells [Bonnet D₁ Dick J.E. Human acute myeloid leukaemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nat. Med. 1997, 3: J30737] which are defined by the expression of CD34 and CD38 surface antigens and have been termed leukaemia stem cells.

Similar tumour-initiating cells have also been found in 'solid' cancers such as prostate [Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ: Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 2005, 65: 1094610951], breast [Al Hajj M, Wicha MS, BenitoHernandez A, Morrison SJ₁ Clarke MF: Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003, 100: 39833988], brain [Singh SK, Hawkins C, Clarke ID, Squire JA₁ Bayani J, Hide T₁ Henkelman RM, Cusimano MD, Dirks PB: Identification of human brain tumour initiating cells. Nature 2004, 432: 396401], lung [Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar L₁ Vogel S, Crowley D, Bronson RT, Jacks T: Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005, 121: 823-835] colon [O'Brien CA, Pollett A, Gallinger S, Dick JE: A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007, 445: 106110; RicciVitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C₁ De Maria R: Identification and expansion of human colon cancer initiating cells. Nature 2007, 445: 111115]; and gastric cancers [Houghton J₁ Stoicov C, Nomura S, Rogers AB, Carlson J₁ Li H, Cai X, Fox JG, Goldenring JR, Wang TC: Gastric cancer originating from bone marrow derived cells. Science 2004, 306: 15681571].

A problem associated with the use of human embryonic stem cells is that it is necessary to sacrifice a human embryo to obtain embryonic stem cell-lines with pluripotent potential. This raises moral issues with respect to isolation of human embryonic stem cells. In relation to cancer stem cells although their isolation does not raise the moral issues associated with human embryonic stem cells a problem exists in relation to the isolation of sufficient numbers of cancer stem cells which can be analysed. For example, in WO2005/089043 is disclosed the isolation of prostate stem cells which have been directly isolated from lymph node and prostate glands from a series of patient samples. The proportion of stem cells in the bulk tumour is around 0.01% and therefore the prostate stem cells are rare and difficult to isolate.

It is known that human somatic cells can be re-programmed to an undifferentiated state similar to an embryonic stem cell. For example, WO2007/069666 describes re- programming of differentiated cells (e.g. mouse fibroblast cells) without the need to use embryonic stem cells. Nuclear re-programming is achieved by transfection of retroviral vectors into somatic cells that encode nuclear re-programming factors, for example Oct family, Sox family, KIf family and Myc family of transcription factors. The somatic cells de-differentiate and express markers of human embryonic stem cells to produce an "induced pluripotent cell" [iPS]. In Takahashi et al [Cell vol 131, p861-872, 2007] adult human dermal fibroblasts with the four transcription factors: Oct3/4, Sox2, Klf4, and c- Myc de-differentiate to human ES cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes and telomerase activity. Furthermore, these iPS cells could differentiate into cell types of the three germ layers in vitro. In a later published manuscript Nakagawa ef al [Published online: 30 November 2007 |doi: 10.1038/nbt1374] show that cMyc can be omitted from the re- programming cocktail and still achieve de-differentiation to a pluripotential state. However, a problem associated with WO2007/069666 and related is the need to transfect the somatic cells with retroviral vectors which result in genome modification. A further example of somatic cell re-programming is described in Yu et a/ [Science vol 318[5858] p1917-1920 2007] which shows that expression of four re-programming factors [Oct 4, Sox2, Nanog and Lin28] in a somatic cells results in cell de-differentiation to a pluripotent state. The iPS cells have a normal karyotype and similarly express ES cell markers and telomerase. Other groups showed that reprogramming human fibroblast to iPSC can be achieved with lentivirus transfection of Oct4, Sox2, Klf4 and UTF1 [Zhao et al Cell Stem Cell 3, p475-479, 2008] or Oct4, Sox2 and SV40 [Mali et al Stem Cells 26(8): 1998-2005, 2008].

The present disclosure relates to the in vitro re-programming of somatic cells that avoids the use of vectors that modify the genome of the recipient cell. Moreover this disclosure also relates to the in vitro re-programming of differentiated cancer cells to a cancer stem cell phenotype.

According to an aspect of the invention there is provided the use of a preparation comprising at least one messenger RNA molecule encoded by a DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in Figure 1 ;

ii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 1 and which encodes a polypeptide with the activity associated with Oct4;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 2;

iv) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 2 and which encodes a polypeptide with the activity associated with Sox2;

v) a DNA molecule comprising a DNA sequence as represented in Figure 3;

vi) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 3 and which encodes a polypeptide with the activity associated with Nanog;

vii) a DNA molecule comprising a DNA sequence as represented in Figure 4;

viii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 4 and which encodes a polypeptide with the activity associated with Klf4;

ix) a DNA molecule comprising a DNA sequence as represented in Figure 5;

x) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 5 and which encodes a polypeptide with the activity associated with cMyc;

xi) a DNA molecule comprising a DNA sequence as represented in Figure 6; xii) a DMA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 6 and which encodes a polypeptide with the activity associated with Lin28;

in the re-programming of a differentiated somatic cell to an induced stem cell.

In a preferred embodiment of the invention said RNA molecule is encoded by a DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in Figure 7;

ii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 7 and which encodes a polypeptide with the activity associated with UTF1 ;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 8;

iv) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 8 and which encodes a polypeptide with the activity associated with SV40 large

T.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular

Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York,

1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65⁰C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65⁰C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

The invention is generally applicable to all mammalian species for example, non-human primates, rodents [e.g. mice, rats, hamsters], cows, sheep, pigs, horses, deer, boar, cats and dogs. In a preferred embodiment of the invention said differentiated somatic cell is human.

In a preferred embodiment of the invention said somatic cell is selected from the group consisting of: a hematopoietic cell, e.g. lymphocyte, myeloid cell; a buccal mucosa cell, an epidermal cell, a mesenchymal cell, a keratinocyte, a hepatocyte e.g. hepatocyte stellate cell.

In a preferred embodiment of the invention said differentiated somatic cell is a fibroblast; preferably an adult or embryonic fibroblast.

In a further preferred embodiment of the invention said differentiated somatic cell is selected from the group consisting of: a dermal fibroblast, a foetal fibroblast, a corneal fibroblast, an intestinal mucosa fibroblast, an oral mucosa fibroblast and urethral fibroblast.

In an alternative preferred embodiment of the invention said stem cell is an induced pluripotent stem cell.

In a further preferred embodiment of the invention said somatic cell is a cancer cell and said induced stem cell is an induced cancer stem cell. As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.

In a preferred embodiment of the invention said cancerous somatic cell is selected from the group consisting of: a cancerous leukaemic cell, a cancerous prostate cell, a cancerous breast cell, a cancerous brain cell, a cancerous lung cell, a cancerous colon cell or a cancerous gastric cell.

In a preferred embodiment of the invention said cancerous somatic cell is a metastatic cancerous cell.

In a preferred embodiment of the invention said preparation comprises mRNA encoding Oct4, Sox2, cMyc and Klf4.

In a further preferred embodiment of the invention said RNA preparation comprises mRNA encoding Oct4, Sox2, Nanog, and Lin28. In a further preferred embodiment of the invention said RNA preparation comprises mRNA encoding Oct4, Sox2, Klf4, cMyc and SV40 LT or Oct4, Sox2, Klf4, UTF1 and SV40 LT.

In a yet further preferred embodiment of the invention said preparation further comprises embryonic stem cell RNA; preferably a polyadenylated mRNA fraction of embryonic stem cell RNA.

According to an aspect of the invention there is provided a method for the re- programming of a differentiated somatic cell to an induced stem cell comprising: i) providing a cell preparation comprising a somatic cell; ii) transfecting said cell preparation with a preparation comprising at least one mRNA that encodes a polypeptide selected from the group consisting of Oct4, Sox2, cMyc.and Klf4,; and iii) culturing said cell preparation and optionally monitoring the differentiation state of said somatic cell.

In a preferred method of the invention said preparation comprises at least one Mrna selected from the group consisting of: Nanog, LIN28, UTF1 and SV40 LT

In a preferred method of the invention said differentiated somatic cell is a fibroblast.

In an alternative preferred method of the invention said stem cell is an induced pluripotent stem cell.

In a further preferred method of the invention said somatic cell is a cancer cell.

In a preferred method of the invention said somatic cell is a metastatic cancer cell.

In a preferred method of the invention said preparation comprises mRNA encoding Oct4, Sox2, cMyc and Klf4.

In a preferred method of the invention said RNA preparation comprises mRNA encoding Oct4, Sox2, Nanog, and Lin28. In a preferred method of the invention said preparation comprises mRNA encoding Oct4, Sox2, cMyc, Klf4 and SV40 LT.

In a still further preferred method of the invention said preparation further comprises embryonic stem cell RNA; preferably a polyadenylated mRNA fraction of embryonic stem cell RNA.

According to a further aspect of the invention there is provided an induced stem cell obtained by the method according to the invention.

In an alternative preferred embodiment of the invention said induced stem cell is an induced pluripotent stem cell.

In a still further preferred embodiment of the invention said induced stem cell is an induced cancer stem cell.

According to a further aspect of the invention there is provided a method for the identification of genes associated with induced stem cells comprising: i) providing a preparation comprising at least one induced stem cell according to the invention; ii) extracting nucleic acid from said cell preparation; iii) contacting said extracted nucleic acid with a nucleic acid array; and iv) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.

Preferably said method includes the additional steps of: i) collating the signal(s) generated by the binding of said nucleic acid to said binding partner; ii) converting the collated signal(s) into a data analysable form; and optionally; iii) providing an output for the analysed data.

In a preferred method of the invention said preparation comprises induced pluripotent stem cells. In a further preferred method of the invention said preparation comprises induced cancer stem cells.

In a further preferred method of the invention said method includes a comparison of the array signal produced between induced normal stem cells and induced cancerous stem cells.

In an alternative preferred method of the invention said method includes a comparison of the array signal produced between a first induced cancer stem cell sample and a second, different induced cancer stem cell sample.

In a preferred method of the invention said induced stem cell sample is derived from a somatic cell sample isolated from a subject.

In a preferred method of the invention said subject is diagnosed with a cancerous condition.

In a further preferred method of the invention said cancerous condition is a metastatic tumour.

According to a further aspect of the invention there is provided a method for the preparation of a library comprising induced stem cell specific gene expression products comprising the steps: i) providing a preparation comprising an induced stem cell according to the invention; ii) extracting nucleic acid from said cell preparation; iii) preparing a cDNA from ribonucleic acid contained in said extracted nucleic acid; and jv) ligating cDNA formed in (iii) into a vector.

In a preferred method of the invention said vector is a phage based vector.

According to a further aspect of the invention there is provided a method to identify agents capable of inhibiting the proliferation and/or motility of induced cancer stem cells comprising: i) providing culture conditions and an induced cancer stem cell according to the invention; ii) adding at least one agent to be tested; and iii) monitoring the antiproliferative activity of the agent with respect to the induced cancer stem cells.

In a preferred method of the invention the inhibitory activity of said agent is tested with respect to the cancerous somatic cell from which said cancerous stem cell is isolated.

According to a further aspect of the invention there is provided non-human animal model for the analysis of the formation of tumours from administered induced cancer stem cell according to the invention.

In a preferred embodiment of the invention said animal is a mouse, preferably an immune compromised mouse. Preferably said mouse is a SCID mouse or an athymic nude mouse.

According to an aspect of the invention there is provided the use of a preparation comprising at least one DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in Figure 1 ;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 2;

v) a DNA molecule comprising a DNA sequence as represented in Figure 3;

vii) a DNA molecule comprising a DNA sequence as represented in Figure 4; viii) a DNA molecule comprising a ONA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 4 and which encodes a polypeptide with the activity associated with Klf4;

ix) a DNA molecule comprising a DNA sequence as represented in Figure 5;

xi) a DNA molecule comprising a DNA sequence as represented in Figure 6;

xii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 6 and which encodes a polypeptide with the activity associated with Lin28; in the re-programming of a cancerous somatic cell to an induced cancer stem cell.

In a preferred embodiment of the invention said DNA molecule is included in an expression vector adapted for expression of said DNA molecule.

Typically said adaptation includes, by example and not by way of limitation, the provision of transcription control sequences (promoter sequences) which mediate ceWtissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only, and not by way of limitation. Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences and is therefore position independent). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues. Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers and autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50kb DNA). Episomal vectors of this type are described in WO98/07876. Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 is the DNA sequence of human Oct4;

Figure 2 is the DNA sequence of human Sox2

Figure 3 is the DNA sequence of human Nanog

Figure 4 is the DNA sequence of human Klf4

Figure 5 is the DNA sequence of human cMyc

Figure 6 is the DNA sequence of human Lin28;

Figure 7 is the DNA sequence of UTF1

Figure 8 is the DNA sequence of SV40 large T

Figure 9 illustrates the electroporation of total RNA from NT2 cells into K562 cells enhances the translation of GFP (green fluorescence protein) mRNA. (a and b), in the absence of GFP mRNA and NT2 total RNA, K562 cells display no green fluorescence, (c and d), electroporation of GFP mRNA into K562 cells leads to the expression of GFP protein which gives green fluorescence, (e and f). co-electroporation of NT2 cell total RNA and GFP mRNA into K562 cells results in enhanced GFP protein expression, a, c and e, bright field images, b, d and f, green channel image. The amount of RNA electroporated is as indicated on the left;

Figure 10 illustrates expression of pluripotency marker genes Oct4 and Nanog in K562 cells following the electroporation of NT2 total RNA or mRNA. 24 hours post electroporation, total RNA was extracted from 1 million K562 cells of following groups and subjected to RT-PCR detection of Oct4 and Nanog. Groups: A. no RNA. B. GFP mRNA. C. GFP mRNA + NT2 total RNA. D. GFP mRNA + NT2 mRNA. Arrows indicate the bands corresponding to the PCR products of Oct4 and Nanog; Figure 11 illustrates increase of Oct4 protein expression 4 days after electroporation. 4 days post electroporation, 1 million K562 cells from following groups (3. no RNA. 4. GFP mRNA. 5. GFP mRNA + NT2 total RNA. 6. GFP mRNA + NT2 mRNA) are lysed and subjected to western blot detection of Oct4 protein. Oct4 protein was detected in 0.1 million of undifferentiated NT2 cells (lane 1), but not in differentiated NT2 cells (lane 2). Low level of Oct4 protein was present in lane 5 and 6, but not lane 3 and 4. Arrow indicates the bands corresponding to Oct4 protein.

Figure 12 illustrates a schematic description of polyA mRNA amplification. A poly dT20 oligo will be used to hybridize to polyA sequence of the mRNA to facilitate the reverse transcription of full-length antisense single-stranded cDNAs. Next, a polyadenlation reaction is carried out. Polyadenlated cDNAs are subjected to one cycle of PCR with an oligo consisted of a T7 promoter at 5¹ end, followed by a poly dT20 sequence with a 3' dNTP clamp. The resulting PCR products are double stranded full-length cDNAs with a T7 promoter and T20 at 5¹ end. Finally, an in vitro transcription reaction is performed using a T7 RNA polymerase. The resulting mRNA products are amplified sense mRNAs of human ES cells.

Figure 13. illustrates western blot of protein expression of reprogramming factors following mRNA electroporation. The mRNA expressed is indicated on the top, the antibodies used is as noted on the left of the figure.

Figure 14. A Showing Oct4-cherry localises to nucleus 4 hours after mRNA microinjeciton into HuF1 cells. B. Examples of alkaline phosphotase positive cell aggregates formation after transfection with mRNAs encoding reprogramming factors and small molecule treatment. The component of mRNA mixture and small molecules are as noted at the bottom.

Figure 15. illustrates quantitative PCR analysis of embryonic stem cell marker genes: Oct4, Nanog and DNMT3b expression in hESC HUES1 cell lines and HuF1 fibroblast cells transfected with reprogramming factors.

Materials and Methods In vitro transcription

To make mRNA in vitro, genes of interest are cloned into RN3P vector (Na J and Zernicka-Goetz M, Current Biology 2006 Jun 20:16(12):1249-54V The vector is first linearized with Sfi I, followed by DNA extraction and precipitation. The linearized DNA is resuspended in ddH2O at 0.5 ug/ul. In vitro transcription reactions are carried out using the Epicentre AmpliCap-Max™ T3 High Yield Message Maker Kit (Cat No. ACM04033) according to manufacturer's instruction. After in vitro transcription, mRNAs are extracted by phenokchloroform, then chloroform, and precipitated by Na Acetate and EtOH method. The mRNA pellet is resuspend in ddH2O at 2 ug/ml and stored in -80 degree until use.

Total cellular RNA and mRNA purification

Cells are lysed in TRIZOL (Invitrogen) and total RNA is purified according to manufacturer's instructions. The total RNA is resuspended in ddH2O and stored in -80 degree until use. The polyA mRNA fraction is purified from the total RNA using Qiagen Oligotex Direct mRNA kit following manufacturer's instructions. The purified polyA mRNA enriched fraction is resuspended in ddH2O and stored in -80 degree.

Electroporation

Human K562 cells, fibroblast cells, keratinocytes and neural stem cells were electroporated on a BTX830 electroporator or Microporator using pre-optimized parameters. Following electroporation, cells were transferred into pre-warmed maintenance medium. Next day, the medium was replaced with MEF conditioned HES medium with small molecules depending on the experiment.

Western blot The following antibodies are used: Oct4 (Santa Cruz, sc-5279), Nanog (R&D systems, AF1997), Sox2 (Chemicon, AB5603), cMyc (Santa Cruz, sc-764), Klf4 (Santa Cruz, SC20691), LIN28 (R&D systems, AF3757),

Cloning of embryonic stem cell specific genes

Oct4, Sox2, cMyc, Klf4, Nanog, Lin28 are cloned using the One-Step RT-PCR" kit from Invitrogen with gene specific primers. The PCR products were ligated into pGEM-Easy vector (Promega) and the sequence were confirmed by sequencing. The coding sequence of each gene was amplified with the second pair of gene specific primers all having EcoR I site and Kozak sequence at 5'end and Not I site at 3' end. The PCR products were cut with EcoRI and Not I and ligated in between the corresponding sites of RN3P vector .

Analysis of reproqrammed pluripotent cells

I. Morphology. Reprog rammed pluripotent stem cells will form compact colonies similar to human ES cells and very different from fibroblast, lymphocytes and mesenchymal stem cells.

II. Gene expression profiles. The expression of pluripotency marker genes in reprogrammed cells will be analysed by RT-PCR and microarray.

III. Alkaline phosphotase assay. The activity of alkaline phosphotase is analysed using the Alkaline Phosphatase Red Microwell Substrate Solution (Sigma AR0100 & AR0200)

IV. Protein expression profile. The expression of ES cell specific transcription factors, Oct4, Sox2, Nanog, will be analysed by western blot as showed in figure 9. Reprogrammed pluripotent cells should express the proteins of above factors at similar levels as undifferentiated ES cells.

V. Surface marker profile. Reprogrammed pluripotent cells should express human ES cell specific surface markers (SSEA3, SSEA4 and TRA1-60) at similar level as undifferentiated cells.

Vl. Differentiation ability. Reprogrammed pluripotent cells will be induced to differentiate in vitro by embryonic body method, and in vivo by cell injection into nude mice. Their ability to differentiate into ectoderm, mesoderm and endoderm tissues will be confirmed by RT-PCR or microarray analysis of gene expression, protein immunostaining and histology studies.

Claims

1 The use of a preparation comprising at least one messenger RNA molecule encoded by a DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in Figure 1 ;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 2;

v) a DNA molecule comprising a DNA sequence as represented in Figure 3;

vii) a DNA molecule comprising a DNA sequence as represented in Figure 4;

viii)a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 4 and which encodes a polypeptide with the activity associated with Klf4;

ix) a DNA molecule comprising a DNA sequence as represented in Figure 5;

xi) a DNA molecule comprising a DNA sequence as represented in Figure 6;

xii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 6 and which encodes a polypeptide with the activity associated with Lin28; in the re-programming of a differentiated somatic cell to an induced stem cell.

2. The use of a preparation comprising at least one messenger RNA molecule encoded by a DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in

Figure 7;

H) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 7 and which encodes a polypeptide with the activity associated with UTF1 ;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 8;

iv) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 8 and which encodes a polypeptide with the activity associated with SV40 large T.

3. Use according to claim 1 or 2 wherein said differentiated somatic cell is human.

4. Use according to any of claims 1-3 wherein said somatic cell is selected from the group consisting of: a hematopoietic cell, e.g. lymphocyte, myeloid cell; a buccal mucosa cell, an epidermal cell, a mesenchymal cell, a keratinocyte, a hepatocyte.

5. Use according to any of claims 1-3 wherein said differentiated somatic cell is an adult fibroblast.

6. Use according to any of claims 1-3 wherein said fibroblast is an embryonic fibroblast.

7. Use according to claim 5 wherein said fibroblast is selected from the group consisting of: a dermal fibroblast, a foetal fibroblast, a corneal fibroblast, an intestinal mucosa fibroblast, an oral mucosa fibroblast and urethral fibroblast.

8. Use according to any of claims 1-7 wherein said stem cell is an induced pluripotent stem cell.

9. Use according to any of claims 1-3 wherein said somatic cell is a cancer cell and said induced stem cell is an induced cancer stem cell.

10. Use according to claim 9 wherein said cancerous somatic cell is selected from the group consisting of: a cancerous leukaemic cell, a cancerous prostate cell, a cancerous breast cell, a cancerous brain cell, a cancerous lung cell, a cancerous colon cell or a cancerous gastric cell.

11. Use according to claim 9 or 10 wherein said cancerous somatic cell is a metastatic cancerous cell.

12. Use according to claim 1 wherein said preparation comprises mRNA encoding Oct4, Sox2, cMyc and Klf4.

13. Use according to claim 1 wherein said RNA preparation comprises mRNA encoding Oct4, Sox2, Nanog, and Lin28.

14. Use according claim 1 wherein said RNA preparation comprises mRNA encoding Oct4, Sox2, Klf4, cMyc and SV40 LT or Oct4, Sox2, Klf4, UTF1 and SV40 LT.

15. Use according to any of claims 1-14 wherein said preparation further comprises embryonic stem cell RNA.

16. Use according to claim 15 wherein said RNA is a polyadenylated mRNA fraction of embryonic stem cell RNA.

17. A method for the re-programming of a differentiated somatic cell to an induced stem cell comprising: i) providing a cell preparation comprising a somatic cell; ii) transfecting said cell preparation with a preparation comprising at least one mRNA that encodes a polypeptide selected from the group consisting of Oct4, Sox2, cMyc.and KIf 4,; and iii) culturing said cell preparation and optionally monitoring the differentiation state of said somatic cell.

18. The method according to claim 17 wherein said preparation comprises at least one mRNA selected from the group consisting of: Nanog, LIN28, UTF1 and SV40 LT

19. The method according to claim 17 or 18 wherein said differentiated somatic cell is a fibroblast.

20. The method according to any of claims 17-19 wherein said stem cell is an induced pluripotent stem cell.

21. The method according to claim 17 or 18 wherein said somatic cell is a cancer cell.

22. The method according to claim 21 wherein said somatic cell is a metastatic cancer cell.

23. The method according to any of claims 17-22 wherein said preparation comprises mRNA encoding Oct4, Sox2, cMyc and Klf4.

24. The method according to any of claims 17-22 wherein said RNA preparation comprises mRNA encoding Oct4, Sox2, Nanog, and Lin28.

25. The method according to any of claims 17-22 wherein said preparation comprises mRNA encoding Oct4, Sox2, cMyc, Klf4 and SV40 LT.

26. The method according to any of claims 17-25 wherein said preparation further comprises embryonic stem cell RNA.

27. The method according to claim 26 wherein said RNA is a polyadenylated mRNA fraction of embryonic stem cell RNA.

28. An induced stem cell obtained by the method according to any of claims 17-27.

29. An induced stem cell according to claim 28 wherein said induced stem cell is an induced pluripotent stem cell.

30. An induced stem cell according to claim 28 wherein said induced stem cell is an induced cancer stem cell.

31. A method for the identification of genes associated with induced stem cells comprising: i) providing a preparation comprising at least one induced stem cell according to any of claims 28-30; ii) extracting nucleic acid from said cell preparation; iii) contacting said extracted nucleic acid with a nucleic acid array; and iv) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.

32. The method according to claim 31 wherein said method includes the additional steps of: i) collating the signal(s) generated by the binding of said nucleic acid to said binding partner; ii) converting the collated signal(s) into a data analysable form; and optionally; iii) providing an output for the analysed data.

33. The method according to claim 31 or 32 wherein said preparation comprises induced pluripotent stem cells.

34. The method according to claim 31 or 32 wherein said preparation comprises induced cancer stem cells.

35. The method according to claim 34 wherein said method includes a comparison of the array signal produced between induced normal stem cells and induced cancerous stem cells.

36. The method according to claim 34 wherein said method includes a comparison of the array signal produced between a first induced cancer stem cell sample and a second, different induced cancer stem cell sample.

37. The method according to claim 31 or 32 wherein said induced stem cell sample is derived from a somatic cell sample isolated from a subject.

38. The method according to claim 37 wherein said subject is diagnosed with a cancerous condition.

39. The method according to claim 38 wherein said cancerous condition is a metastatic tumour.

40. A method for the preparation of a library comprising induced stem cell specific gene expression products comprising the steps: i) providing a preparation comprising an induced stem cell according to any of claims 28-30; ii) extracting nucleic acid from said cell preparation; iii) preparing a cDNA from ribonucleic acid contained in said extracted nucleic acid; and iv) ligating cDNA formed in (iii) into a vector.

41. The method according to claim 40 wherein said vector is a phage based vector.

42. A method to identify agents capable of inhibiting the proliferation and/or motility of induced cancer stem cells comprising: i) providing culture conditions and an induced cancer stem cell according to any of claims 28-30; ii) adding at least one agent to be tested; and iii) monitoring the anti-proliferative activity of the agent with respect to the induced cancer stem cells.

43 The method according to claim 42 wherein the inhibitory activity of said agent is tested with respect to the cancerous somatic cell from which said cancerous stem cell is isolated.

44. A non-human animal model for the analysis of the formation of tumours from administered induced cancer stem cell according to any of claims 28-30.

45. A non-human animal model according to claim 44 wherein said animal is a mouse.

46. A non-human animal according to claim 45 wherein said mouse is an immune compromised mouse.

47. The use of a preparation comprising at least one DNA molecule selected from the group consisting of:

i) a DNA molecule comprising a DNA sequence as represented in Figure 1;

iii) a DNA molecule comprising a DNA sequence as represented in Figure 2;

iv) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in

Figure 2 and which encodes a polypeptide with the activity associated with Sox2;

v) a DNA molecule comprising a DNA sequence as represented in Figure 3;

vi) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 3 and which encodes a polypeptide with the activity associated with Nanog; vii) a DNA molecule comprising a DNA sequence as represented in Figure 4;

viii) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in Figure 4 and which encodes a polypeptide with the activity associated with KIf 4;

ix) a DNA molecule comprising a DNA sequence as represented in Figure 5;

x) a DNA molecule comprising a DNA sequence that hybridizes under stringent hybridization conditions to the DNA sequence in

Figure 5 and which encodes a polypeptide with the activity associated with cMyc;

xi) a DNA molecule comprising a DNA sequence as represented in Figure 6;

48. Use according to claim 47 wherein said DNA molecule is included in an expression vector adapted for expression of said DNA molecule.