Patent Application
20100093090
This application claims the benefit of Chinese Patent Application No. 200810091841.2, filed Apr. 3, 2008, which is hereby expressly incorporated by reference in its entirety.
The invention relates to the conversion of a differentiated somatic cell into a naive cell, e.g., stem cell such as an induced pluripotent stem cell.
It is reported that a somatic cell could be reprogrammed to be a stem cell, which is more naive and have more potential of self-renewal and differentiation. This reprogramming process could be induced by nuclear transplantation into Oocytes (Campbell, McWhir et al. 1996; Jouneau and Renard 2003), by fusion with pluripotent stem cells (Cowan, Atienza et al. 2005), by contacting with chemical compounds, or induced by culture methods (Chen, Zhang et al. 2004).
In August, 2006, Yamanaka's lab reported that mouse embryonic fibroblast cells could be reprogrammed to be “induced pluripotent stem cells (iPS cells)”, through retro-viral introduction of four transcriptional factors, Oct4, Sox2, Klf4 and c-Myc (Takahashi and Yamanaka 2006). After that, Oct4, Sox2, Klf4 and c-Myc were so-called reprogramming factors. In November 2007, Yamanaka's lab and Thomson's lab both reported the generation of human iPS cells from adult human fibroblasts by the combination “Oct4, Sox2, Klf4 and c-Myc” or “Oct4, Sox2, Nanog, Lin-28”, respectively (Takahashi, Tanabe et al. 2007; Yu, Vodyanik et al. 2007). IPS cells were very similar to embryonic stem cells in gene profiling, differentiation potential and epigenetic modifications (Maherali, Sridharan et al. 2007). They were able to self-renew and differentiate into all mature cell types, including neurons, hematopoietic cells, muscle cells and islet cells.
Further study found that c-myc was not necessary in this combination. iPS cells could be induced by Oct4, Sox2 and Klf4, without c-myc although the efficiency was much lower (Nakagawa, Koyanagi et al. 2008; Wernig, Meissner et al. 2008). Further study showed that, Sox2, Klf4 and c-myc could also be substituted by other members in their gene family. For examples, Sox2 could be substituted by Sox1 or Sox3; Klf4 could be substituted by KLF2 and KLF5; while, c-myc could be substituted by L-myc and N-myc (Nakagawa, Koyanagi et al. 2008). Other genes were identified to improve the reprogramming efficiency or replace some of the four transcriptional factors in this combination. For example, Esrrb could substitute Klf4 to induce iPS cells with Oct4 and Sox2 (Feng, Jiang et al. 2009); SV40 T could greatly enhance reprogramming efficiency (Mali, Ye et al. 2008). We found that p53 inactivation factor and UTF1 could greatly improve iPS cell generation efficiency (Zhao, Yin et al. 2008). After that, six groups reported that p53 pathway was a major barrier of iPS cell generation (Banito, Rashid et al. 2009; Hong, Takahashi et al. 2009; Kawamura, Suzuki et al. 2009; Li, Collado et al. 2009; Marion, Strati et al. 2009; Utikal, Polo et al. 2009).
Particularly, several chemical compounds were reported to be involved in reprogramming process. VPA (an HDAC inhibitor) and 5-aza-C (a DNA methyl transferase inhibitor) were reported to improve reprogramming efficiency (Huangfu, Maehr et al. 2008; Mikkelsen, Hanna et al. 2008). CHIR 99021 (a GSK3 inhibitor) and PD0325901 (an MEK/ERK inhibitor) could help push partial reprogrammed cells into fully reprogrammed iPS cells (Silva, Barrandon et al. 2008). Furthermore, BIX01294 were reported to replace oct4 to induce iPS cells from NSCs (Shi, Do et al. 2008). VPA were also reported to replace KLF4 and induce reprogramming with only Oct4 and Sox2 from human foreskin fibroblasts (Huangfu, Osafune et al. 2008). BIX01294 and BAYK 8644 were reported to induce iPS cells with only Oct4 and Klf4 (Shi, Desponts et al. 2008). Kenpaullone could substitute KLF4 and induce iPS cells with Oct4, sox2 and c-myc (Lyssiotis, Foreman et al. 2009).
There were cell types more amenable to reprogramming. For example, human keratinocytes and amniotic fluid-derived cells were reported to be reprogrammed by a much higher efficiency (Aasen, Raya et al. 2008; Li, Zhou et al. 2009). NSCs were similar to ESCs (or iPSCs) in gene profiling, and express Sox2, c-myc and Klf4. They could be induced into iPS cells with only one transcriptional factor, Oct4, both in mouse and human (Kim, Greber et al. 2009; Kim, Sebastiano et al. 2009). Melanocytes were also reported to be induced to iPS cells without sox2, although there was no evidence to their expression of sox2 (Utikal, Maherali et al. 2009). Moreover, somatic cells from patients were also reported to be induced to iPS cells for potential clinical applications (Dimos, Rodolfa et al. 2008; Park, Arora et al. 2008; Raya, Rodriguez-Piza et al. 2009).
Furthermore, iPS cells could be induced not limited to retro-viral infection. DNA transduction, adenoviral delivery or protein delivery of the four reprogramming factors could also be applied in iPS cell induction (Okita, Nakagawa et al. 2008; Stadtfeld, Nagaya et al. 2008; Kim, Kim et al. 2009; Zhou, Wu et al. 2009; Zhou and Freed 2009). Moreover, the four reprogramming factors could also be conjunct in a single polycistronic vector to induce iPS cells (Carey, Markoulaki et al. 2009; Sommer, Stadtfeld et al. 2009).
The methods described herein can be used, for example, to optimize (e.g., improve speed or efficiency) the creation of induced cells, e.g., induced pluripotent stem (iPS) cells from other cell types (e.g., an adult cell and/or a somatic cell), including, but not limited to the creation of iPS cells from human biopsies, such as blood, skin, fat, hair follicle, mucus, etc. The iPS cell lines so created can be used to study differentiation and disease mechanisms/pathology.
In one aspect, the invention features a method for reprogramming a somatic cell, wherein the method comprises treating the somatic cell with factor A selected from one or more of the group consisting of Oct4, Klf4, Sox2, c-Myc, a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and a DNA methyl transferase inhibitor, and factor B selected from one or two of the group consisting of UTF1 and a p53 inactivation factor, or the combinations thereof.
In a preferable embodiment, the MEK/ERK inhibitor is PD0325901, the HDAC inhibitor is VPA, the GSK3 inhibitor is CHIR 99021, and the DNA methyl transferase inhibitor is 5-aza-C.
In one aspect, the invention features a method of reprogramming an iPS cell from a somatic cell, the method comprising: introducing factor A and factor B into a somatic cell under conditions sufficient to produce an iPS cell from a somatic cell, wherein factor A is selected from one or more (e.g. two, three, four, five, six, seven, eight, nine, or ten) of the group consisting of Oct4, Klf4, Sox2, c-Myc, a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and a DNA methyl transferase inhibitor, and factor B is selected from one or two of the group consisting of UTF1 and a p53 inactivation factor, or the combinations thereof.
In some embodiments, factor A selected from one or more of the group consisting of Oct4, Klf4, Sox2, and c-Myc and/or factor B are in the form of DNA, mRNA or proteins.
In some embodiments, the step of treating the somatic cell with factor A selected from one or more of the group consisting of Oct4, Klf4, Sox2 and c-Myc and factor B selected from one or two of the group consisting of UTF1 and a p53 inactivation factor comprises treating the somatic cell with one heterologous nucleic acid sequence encoding said factor A and said factor B, or the respective heterologous nucleic acid sequences encoding Oct4, Klf4, Sox2, c-Myc, UTF1 or a p53 inactivation factor, or the combinations thereof.
In some embodiments, the somatic cell is treated with the heterologous nucleic acid sequence by infection.
In some embodiments, a p53 inactivated factor is a double strand small RNA and the DNA sequence encoding this small interference RNA is GACTCCAGTGGTAATCTACT.
In some embodiments, a p53 inactivation factor selectively inhibits p53 gene expression.
In some embodiments, a p53 inactivation factor selectively inhibits p53 protein modification, and subsequently impairs p53 protein function.
In some embodiments, a p53 inactivation factor comprises at least two p53 siRNA, or a combination thereof.
In some embodiments, a p53 inactivation factor is a small molecule selectively inhibits p53 mediated signaling pathway. For example, KU55933, which is a p53 pathway inhibitor, which selectively and competitively inhibiting ATM kinase, p53 activity and p21 expression.
In some embodiments, the somatic cell is treated by CHIR 99021 or other chemical compounds reported to improve reprogramming efficiency. In a preferable embodiment, said chemical compounds reported to improve reprogramming efficiency is a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and a DNA methyl transferase inhibitor. In a preferable embodiment, the MEK/ERK inhibitor and the GSK3 inhibitor is as shown above, the DNA methyl transferase inhibitor is as shown below, and HDAC inhibitor is as shown below.
In some embodiments, the method comprises treating the somatic cell with two transcription factors.
In some embodiments, the transcription factors comprise Oct4 and Sox2.
In some embodiments, the method comprises treating the somatic cell with three transcription factors.
In some embodiments, the transcription factors comprise Oct4, Sox2 and Klf4.
In some embodiments, the method comprises treating the somatic cell with four transcription factors.
In some embodiments, the transcription factors comprise Oct4, Sox2, Klf4 and c-Myc.
In some embodiments, the somatic cell is reprogrammed partially or completely. In a preferable embodiment, the somatic cell is reprogrammed completely into the induced pluripotent stem cell.
In some embodiments, the iPS cell has a normal karyotype.
In some embodiments, the somatic cell is a fibroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a liver cell, a GI tract cell, a mammary cell, a hepatocyte, a stomach cell, a keratinocyte cell, a kidney cell, a blood cell, a vascular cells, a skin cell, an immune system cell, a lung cell, a bone cell or a pancreatic islet cell.
In some embodiments, the somatic cell is a primary cell or is a progeny of a primary or secondary cell.
In some embodiments, the somatic cell is derived from mammal. In a preferable embodiment, the mammal is human, murine, or primate.
In some embodiments, the somatic cell is obtained from a sample selected from a group consisting of a hair follicle, a blood sample, a swab sample or an adipose biopsy.
In some embodiments, the somatic cell is a healthy cell or a cell containing at least one genetic lesion.
In some embodiments, a plurality of the iPS cells are produced from a plurality of the somatic cells.
In some embodiments, the method further comprises implanting the iPS cells into a subject.
In some embodiments, the subject is suffering from a disorder. In a preferable embodiment, the disorder is selected from a group consisting of hematopoietic conditions (e.g., sickle cell anemia, leukemias, immune deficiencies), cardiac disorders (e.g., myocardial infarcts, and myopathies) and disorders such as liver disease, diabetes, thyroid abnormalities, neurodegenerative/neurological disorders (e.g., Parkinson's, Alzheimer's, stroke injuries, spinal chord injuries), circulatory disorders, respiratory disorders, wound healing and/or repair, bone repair, and enzyme abnormalities.
In some embodiments, the iPS cells are from a donor different than the subject (e.g., a relative of the subject).
In another aspect, the present invention features a method for improving the reprogramming efficiency of a somatic cell, the method comprising treating the somatic cell with factor B selected from one or two of the group consisting of UTF1 and a p53 inactivation factor.
In still another aspect, the present invention features a method for preparing a medicament for treating disease, characterized in that the usage of the cell produced by the method of claim 1, wherein the disease is selected from the group consisting of hematopoietic conditions, cardiac disorders and disorders such as liver disease, diabetes, thyroid abnormalities, neurodegenerative/neurological disorders, circulatory disorders, respiratory disorders, wound healing and/or repair, bone repair, and enzyme abnormalities.
In a preferable embodiment of all of the above aspects, the somatic cell is treated with a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and/or a DNA methyl transferase inhibitor before/after the treatment of the somatic cell with one or more of the factor selected from the group consisting of Oct4, Klf4, Sox2, c-Myc, UTF1 and a p53 inactivation factor.
In another aspect, the invention features an iPS cell produced by the method described herein.
In one aspect, the invention features a cell expressing Oct4, Sox2, Klf4, c-Myc and UTF1.
In another aspect, the invention features a cell expressing Oct4, Sox2, Klf4, c-Myc and expressing low p53.
In one aspect, the invention features a cell expressing Oct4, Sox2, Klf4 and UTF1.
In one aspect, the invention features a cell expressing Oct4, Sox2, Klf4, and low expressing p53.
In one aspect, the invention features a cell expressing Oct4, Sox2 and UTF1.
In one aspect, the invention features a cell expressing KLF4, and UTF1.
In one aspect, the invention features a cell expressing Oct4, Sox2, and low expressing p53.
In one aspect, the invention features a cell expressing KLF4, and low expressing p53.
In one aspect, the invention features a reaction mixture comprising a more primitive precursor or a less differentiated cell, e.g., a pluripotent stem cell (or a population thereof) compared to a somatic cell from which it was derived and expressed exogenous UTF1 and/or a p53 inactivation factor.
In some embodiments, the less differentiated cell is an iPS cell.
In some embodiments, the iPS cell is produced by a method described herein.
In one aspect, the invention features a composition comprising a cell produced by a method described herein.
In another aspect, the present invention features a kit for reprogramming a somatic cell, comprising factor A selected from one or more (for example, two, three, four, five, six, seven, eight, nine or ten) of the group consisting of Oct4, Klf4, Sox2, c-Myc, a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and a DNA methyl transferase inhibitor, as well as factor B selected from one or two of the group consisting of UTF1 and a p53 inactivation factor.
In a preferable embodiment, the MEK/ERK inhibitor is PD0325901, the HDAC inhibitor is VPA, the GSK3 inhibitor is CHIR 99021, and the DNA methyl transferase inhibitor is 5-aza-C.
In still another aspect, the present invention features a kit comprising: an iPS cell produced by a method of claim 1; at least one component for directing the iPS cell to a differentiated cell or expanding the iPS cell; and instructions.
In a preferable embodiment, the iPS cell is frozen or in culture.
In some embodiments, the p53 inactivated factor and the somatic cell are as described above. In some embodiments, the kit further comprises a component for the detection of a marker for an iPS cell selected from a group consisting of alkaline phophatase, NANOG, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81. Those skilled in the art can determine the component for the detection of a marker based on the knowledge in the art. In some embodiments, the expression of a marker selected from a group consisting of alkaline phophatase, NANOG, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81 is upregulated to by a statistically significant amount in the iPS cell relative to the somatic cell.
In some embodiments, the kit further comprises an iPS cell wherein the iPS cell is produced from the same cell type of the somatic cell by the method described herein.
In some embodiments, the kit further comprises a component for preparation of a karyotype from a cell.
In another aspect, the invention features a kit comprising an iPS cell produced by the method described herein. In a preferable embodiment, the p53 inactivated factor is as described above.
In some embodiments, the iPS cell is an isolated iPS cell.
In some embodiments, the iPS cell is frozen or in culture.
In yet another aspect, the invention features a kit comprising: an iPS cell produced by the method described herein; at least one component for directing the iPS cell to a differentiated cell; and instructions for directing the iPS cell to a differentiated cell.
In some embodiments, the iPS cell is an isolated iPS cell.
In some embodiments, the iPS cell is frozen or in culture.
In some embodiments, the differentiated cell comprises a fibroblast (e.g., primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a liver cell, a GI tract cell, a mammary cell, a kidney cell, a blood cell, a vascular cell, a skin cell, an immune system cell, a lung cell, a bone cell, keratinocyte, or a pancreatic islet cell.
In one aspect, the invention features a kit comprising: an iPS cell produced by the method described herein; at least one component for expanding the iPS cell; and instructions for expanding the iPS cell.
In some embodiments, the iPS cell is an isolated iPS cell.
In some embodiments, the iPS cell is frozen or in culture.
In one aspect, the invention features a method of instructing an end-user to produce an iPS cell from a somatic cell, the method comprises providing the components of a kit for producing an iPS cell from a somatic cell described herein; and instructing the end-user using an information material, e.g., a printed material or a computer readable material, or both.
Accordingly, in one aspect, the disclosure features a method of producing a more primitive precursor or a less differentiated cell, e.g., pluripotent stem cell (or a population thereof) from a somatic cell, or reprogramming a somatic cell. The method comprises: introducing UTF1 or a p53 inactivation factor, or their combination thereof, thereby produce a primitive precursor or a less differentiated cell, e.g., pluripotent stem cell (or a population thereof) from the somatic cell or to reprogram the somatic cell.
In one embodiment of any one of above aspects, the somatic cell further expresses, or has increased expression, of one or more transcription factor(s) (e.g., two, three, or four transcription factors). In one embodiment, the transcription factor is one or more of Oct4, Klf4, Sox2 and c-Myc. In one embodiment, the somatic cell does not express c-Myc or does not express c-Myc at statistically significant levels or does not over express c-Myc.
In one embodiment of any one of above aspects, the somatic cell does not express c-Myc or Klf4 or does not express c-Myc or Klf4 at statistically significant levels or does not over express c-Myc and Klf4. In some embodiments, the somatic cell can express, e.g., Oct4 and Sox2 or the somatic cell can express, e.g, Oct4, Klf4 and Sox2 or the somatic cell can express Oct4, Klf4, Sox2 and c-Myc.
In one embodiment of any one of above aspects, the somatic cell includes a heterologous nucleic acid sequence, e.g., a heterologous nucleic acid sequence encoding transcription factor(s), e.g., a nucleic acid encoding transcription factor(s) described herein. In one embodiment, the nucleic acid encodes Oct4, Klf4, Sox2 or c-Myc. In one embodiment, the somatic cell includes one or more heterologous nucleic acid sequences, e.g., encoding transcription factors, e.g., encoding one or more of Oct4, Klf4, Sox2 and c-Myc. In one embodiment, the somatic cell includes at least three heterologous nucleic acid sequences, e.g., encoding Oct4, Klf4 and Sox2. In one embodiment, somatic cell includes at least two heterologous nucleic acid sequences, e.g., encoding Oct4 and Sox2. In another embodiment, the somatic cell includes at least four heterologous nucleic acid sequences, e.g., encoding Oct4, Klf4, Sox2 and c-Myc. In one embodiment, the nucleic acid sequence is introduced into the somatic cell, or the somatic cell is the progeny of such a somatic cell. In an embodiment the cell does not include a heterologous c-Myc gene. In an embodiment the cell does not include heterologous c-Myc and Klf4 genes. In an embodiment the somatic cell is human and any heterologous gene, e.g., transcription factor gene, is human as well, e.g., the human equivalent of any of Oct4, Klf4, Sox2 and c-Myc. In an embodiment the method includes the further step of selecting a more primitive precursor or a less differentiated cell, e.g., pluripotent stem cell (or a population thereof) made by the method which has lost a vector which encodes the heterologous nucleic acid.
In one embodiment, the somatic cell is as described above.
In an embodiment, the somatic cell is obtained from a first individual and the more primitive precursor or a less differentiated cell, e.g., pluripotent stem cell (or a population thereof) (or a tissue derived therefrom) is administered to the same first individual, or to a second individual, e.g., an individual related to said first individual. The second individual can be an individual who carries a different allele for a selected gene than does the first individual. E.g., the first individual can have an allele which does not cause a disease state or unwanted condition and the second individual has the allele which causes the disease state or unwanted condition.
In one embodiment, the number of stem cells produced, e.g., in the presence of UTF1 or a p53 inactivation factor, or a combination thereof, is 5-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 50-, 100-, 120-, 130-, 140-, 150-, 200-, 250-, 500-, 750- or 1000-fold greater than the number of stem cells produced without UTF1 and/or a p53 inactivation factor.
In another aspect, the disclosure features a population of cells, e.g., pluripotent stem cell or a population of pluripotent stem cells, produced by a method described herein.
In another aspect, the invention features a reaction mixture including a somatic cell and a sufficient amount of Oct4, Klf4, Sox2, c-Myc, a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor, a DNA methyl transferase inhibitor, UTF1 and a p53 inactivation factor, or a combination thereof, to convert the somatic cell to a more primitive precursor or a less differentiated cell, e.g., pluripotent stem cell (or a population thereof). In one embodiment, the somatic cell is treated with one or more transcription factors, for example, a transcription factor selected from Oct4, Klf4, Sox2 and c-Myc. In some embodiments, the somatic cell is treated with one, two, three or four transcription factors (e.g., the somatic cell is treated with Oct4□the somatic cell is treated with Oct4 and Sox2, the somatic cell is treated with Oct4, Sox2, and Klf4 or the somatic cell is treated with Oct4, Sox2, Klf4, and c-Myc). In some embodiments, the somatic cell is not treated with c-Myc and/or Klf4.
In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a cell, e.g., a pluripotent stem cell or a population of pluripotent stem cells, produced by a method described herein.
The methods and pluripotent stem cells described herein are useful for treating a wide variety of conditions, including hematopoietic conditions (e.g., sickle cell anemia, leukemias, immune deficiencies), cardiac disorders (e.g., myocardial infarcts, and myopathies) and disorders such as liver disease, diabetes, thyroid abnormalities, neurodegenerative/neurological disorders (e.g., Parkinson's, Alzheimer's, stroke injuries, spinal chord injuries), circulatory disorders, respiratory disorders, wound healing and/or repair, bone repair, and enzyme abnormalities.
In one embodiment, the disclosure features a method of treating a disorder described herein, wherein the method includes: administering a pluripotent stem cell or a population of pluripotent stem cells produced by a method described herein to a subject, e.g., a subject that suffers from a disorder described herein.
In one embodiment of the methods described herein, the somatic cell contains one or more genetic defect, and, e.g., the pluripotent stem cell produced by a method described herein includes the genetic defect or defects. In some embodiments, the genetic defect is corrected (e.g., by homologous recombination) in the pluripotent stem cell, e.g., to provide a corrected pluripotent stem cell. Such cells can be administered by known methods such as the methods described e.g., in U.S. Publication No: 20030228293, the contents of which is incorporated herein by reference. The genetic defect corrected can be, for example, a genetic defect that causes an immune system disorder; a genetic defect that causes a neurological disorder; a genetic defect that causes a cardiac disorder; a genetic defect that causes a circulatory disorder; a genetic defect that causes a metabolic disorder such as diabetes; or a genetic defect that causes a respiratory disorder.
In some embodiments of the methods described herein, the pluripotent stem cell or population of pluripotent stem cells are differentiated in vitro into tissue or cell types useful in treating the condition or disorder described herein. In one embodiment, the pluripotent stem cell or tissues or cell types derived from the pluripotent stem cells are introduced into the subject from which the somatic cell was obtained. In one embodiment, the somatic cell is obtained from a subject having one or more genetic defects and the corrected pluripotent stem cell or a tissue of cell type derived from the corrected pluripotent stem cell is reintroduced to the subject. Differentiation can be effected by known methods. In one embodiment, the pluripotent stem cells are used to produce hematopoietic stem cells (HSC) which are, e.g., useful for transplantation and restoration of immune function in immune deficient recipients.
The methods described herein can further include maintaining the pluripotent stem cells under conditions which result in their differentiation into a desired cell type(s) (e.g., into repaired neurons, cardiac myocytes, blood cell type, bone cell (e.g., osteoblast) or pancreatic cells).
In one aspect, the invention includes a method for manufacturing a medicament for treating a disorder described herein, comprising providing a cell (e.g., an iPSC) produced by the method described herein. The medicament can include other features described herein.
Kits for practicing the methods disclosed herein and for making cells disclosed herein (e.g., iPS cells) are included.
In one aspect, a kit will contain a somatic cell, the components described herein (e.g., a p53 inactivation factor etc.) used to converting the somatic cell to an iPS cell and instructions for converting a somatic cell to an iPS cell using the method described herein.
In one embodiment, the somatic cell is directed to an iPS cell. In one embodiment, the somatic cell can be used as a control.
In another aspect, the invention features a kit comprising an iPS cell made by a method described herein and one or more component(s) for expanding (e.g., multiplying or proliferating) the iPS cell. The component(s) for expanding (e.g., multiplying or proliferating) the iPS cell are known in the art.
In another aspect, a kit contains an iPS cell, for example, made by a method described herein and instructions for directing an iPS cell to a differentiated cell.
The kit will typically be provided with its various elements included in one package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g., a styrofoam box. The enclosure can be configured so as to maintain a temperature differential between the interior and the exterior, e.g., it can provide insulating properties to keep the reagents at a preselected temperature for a preselected time.
The kit can include one or more containers for the composition containing a component(s) described herein.
In one aspect, the invention features a method for reprogramming a somatic cell to form a less differentiated cell comprising treating factor A and factor B into a somatic cell, wherein factor A is selected from one or more of the group consisting of Oct4, Klf4, Sox2, c-Myc, a MEK/ERK inhibitor, BIX01294, BAYK 8644, Kenpaullone, a GSK3 inhibitor, an HDAC inhibitor and a DNA methyl transferase inhibitor, and factor B is selected from one or two of the group consisting of UTF1 and a p53 inactivation factor, thereby producing a cell that is less differentiated than the somatic cell (e.g., an ES-like cell).
In yet another aspect, the invention features a reprogrammed somatic cell produced by the method described herein, in which expression of a plurality of genes that are up-regulated or down-regulated in ES cells is up- or down-regulated in the reprogrammed somatic cell, wherein these genes are not up- or down-regulated in the somatic cell prior to reprogramming.
In some embodiments, expression of the genes Rex3 and Zfp7 is up-regulated, and expression of the genes Aspn and Meox2 are down-regulated compared to the expression of these genes in the somatic cell prior to reprogramming.
In one aspect, the invention features a reprogrammed somatic cell in which expression of a plurality of genes that are specifically expressed in ES cells are up-regulated in the reprogrammed somatic cell, wherein these genes are not up-regulated in the somatic cell prior to reprogramming.
In another aspect, the invention features a reprogrammed somatic cell produced by the method described herein, in which expression of a plurality of genes that are specifically expressed in the somatic cells prior to reprogramming, but are not expressed in ES cells, are down-regulated in the reprogrammed somatic cell, wherein these genes are not down-regulated in the somatic cell prior to reprogramming.
In yet another aspect, the invention features a reaction mixture comprising a somatic cell and (i) UTF1, (ii) a p53 inactivation factor, or (iii) a mixture thereof.
As described herein, UTF1 and a p53 inactivation factor can be employed to efficiently generate induced pluripotent stem (iPS) cells from skin fibroblasts or other cell types.
iPS cells can be created by over-expression of one or more genes, for example one or more of the following four genes: Oct4, Sox2, c-Myc and Klf4 through retroviral infection, but with low efficiencies. All of these four genes are known to be or considered to be DNA binding proteins, transcription factors. Notably, the oncogene c-Myc used in this approach causes tumor formation in cells derived from the iPS cells. Although iPS cells can be generated with only Oct4, Sox2 and Klf4, the efficiency is even lower; fewer than iPS colonies from out of 100,000 cells. These issues pose significant barriers for creation of iPS cells for therapeutic applications. One obvious concerns is the use of retroviruses, which integrate into chromosomal DNA and can cause ancillary problems (mutations). Beyond the use of retroviral vectors and the insertion mutations they cause, the methods involves adding new reprogramming factors (eg. new transcriptional factors or new chemical compounds) to the cell.
As described herein, additional reprogramming factors such as Utf1 and/or a p53 inactivation factor can improve the efficiency of iPS cell induction up to more than 100 fold. For example, infection with Utf1 and p53 siRNA, induced 0.1-0.2% iPS cells in human fibroblasts infected with the four factors (Oct4, Sox2, c-Myc and Klf4), a more than 100 fold improvement over the non-treated control (−0.001%). Infection with p53 siRNA and Utf1 induced 0.1-0.2% iPS cells in human fibroblasts infected with the three factors (Oct4, Sox2 and Klf4, but not c-Myc).
As described herein, infection of a p53 inactivation factor (such as p53 siRNA) and Utf1 enables reprogramming of human cells by only three other transcription factors, Oct4 and Sox2 and Klf4, without the need for the oncogenes c-Myc. For example, iPS colonies were identified about 1 month post-infection in human fibroblasts infected by Oct4, Sox2 and Klf4 together with p53 siRNA and Utf1. On average, about 71-223 iPS cells were successfully obtained out of every 100,000 human adult fibroblast cells after infection.
Moreover, infection of p53 siRNA or Utf1 enables reprogramming of human cells by only three other transcription factors, Oct4 and Sox2 and Klf4, without the need for the oncogenes c-Myc. For example, iPS colonies were identified about 1 month post-infection in human fibroblasts infected by Oct4, Sox2 and Klf4 together with p53 siRNA or Utf1. On average, about 30 iPS cells were successfully obtained out of every 100,000 human adult fibroblast cells after infection.
The methods described herein improve the efficiency of creating iPS cells from skin (e.g., human skin cells) and are useful for making induced stem cells from other cell types with or without using the oncogenes c-Myc. For example, these additional factors may make it possible to create iPS cells from small numbers of cells (e.g., such as those obtained from hair follicle cells from patients, blood samples, adipose biopsy, etc), something that could otherwise be difficult or impossible due to the low efficiency of the current method. Thus, the addition of genes (e.g. p53 inactivated factor and/or Utf1 etc.) or small molecules compounds (e.g., chemicals described herein) can increase the probability of success when trying to make iPS cells from human skin biopsies (fibroblasts or other nucleated cells) and may be helpful in creating iPS cells from any other cell types.
Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. The two broad types of mammalian stem cells are: embryonic stem cells that are found in blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.
Stem cells can be used, e.g., in bone marrow transplants to treat leukemia. Stem cells can be used to treat diseases including cancer, Parkinson's disease, muscle damage, burns, heart disease, diabetes, osteoarthritis, rheumatoid arthritis, hematopoietic conditions (e.g., sickle cell anemia, leukemia, lymphoma, inherited blood disorders), immune deficiencies), cardiac disorders (e.g., myocardial infarcts, and myopathies) and disorders such as liver disease, diabetes, thyroid abnormalities; neurodegenerative/neurological disorders (e.g., Parkinson's Disease, Alzheimer's Disease, stroke injuries, spinal chord injuries), Crohn's Disease, circulatory disorders, respiratory disorders, wound healing and/or repair, bone repair, and enzyme abnormalities.
The methods described herein can be used, e.g., to reprogram somatic cells to a pluripotent state. Such somatic cells can be obtained, for example from a patient, to prepare patient-specific stem cells (e.g., patient-specific pluripotent stem cells). A variety of cells can be used, such as, hair follicle cells, a cell from a blood sample, a cell from adipose tissue, a stomach cell, a liver cell, or a cell from skin (e.g., fibroblast or other cell type, e.g., keratinocyte, melanocyte, Langerhans cell, or Merkel cell) and so on.
Somatic cells are any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as gametes) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells—is a somatic cell. For example, internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells.
Additional cell types include: a fibroblast (e.g., aprimary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammary cell, a hepatocyte and a pancreatic islet cell. In some embodiments, the somatic cell is a primary cell line or is the progeny of a primary or secondary cell line. In one embodiment, the somatic cell is obtained from a sample, e.g., a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g., an oral swab sample).
The p53 tumour suppressor protein is a gatekeeper of cellular fate in multicellular organisms. p53 is activated in response to genotoxic stress and initiates cell cycle arrest, senescence and/or apoptosis via pathways involving transactivation of p53 target genes. p53-controlled transactivation of target genes is an essential feature of each stress response pathway, although some effects of p53 may be independent of transcription. As a transcription factor that both activates and represses a broad range of target genes, p53 demands an exquisitely complicated network to control and fine-tune responses to the various stress signals encountered by cells. p53 contributed to spontaneous and DNA damage-induced apoptosis of hESCs through a transcription-independent mitochondrial pathway.
Examples of p53 inactivation factors include Pifithrin-α, Pifithrin-α Cyclic-, Pifithrin-α p-Nitro-, Pifithrin-α p-Nitro-Cyclic-, ATM/ATR Kinase Inhibitor, Caspase Inhibitor VI, ATM Kinase Inhibitor, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), CHL (Chlorophyllin), RTK1 (plumbagin), Dicoumarol, ATO (Arsenic trioxide), cyclosporin A, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), Bax-Inhibiting Peptide (V5), Bax inhibitor peptide P5, Chk2 Inhibitor, Chk2 Inhibitor II, Debromohymenialdisine, Stylotella aurantium, hydrobromide, CGK 733. Other inhibitors include, for example, dominant negative forms of the p53 (e.g., catalytically inactive forms) siRNA inhibitors of p53, and antibodies that specifically bind to p53. Inhibitors are available, e.g., from Tocris, Calbiochem and Sigma Aldrich.
The UTF1 (undifferentiated embryonic cell transcription factor 1) gene, is specifically expressed in the inner cell mass and primitive ectoderm and is down-regulated at early primitive streak stages. Expression is maintained in the primordial germ cells in developing embryos and in the gonads in adult animals. Promoter analysis indicated that the murine UTF1 gene is transcriptionally regulated by Oct4 and Sox2. The UTF1 protein was shown to repress transcription, to activate reporter genes in an ATF2-dependent manner, and to interact with the basal transcription factor TFIID. A recent study suggested a role for UTF1 in the proliferation rate and teratoma-forming capacity of ES cells. Human UTF1 is a tightly DNA-associated protein with transcriptional repressor activity, as a pluripotency-associated chromatin component with core histone-like characteristics.
Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from an [epsilon]-N-acetyl lysine amino acid on a histone. Exemplary HDACs include those Class I HDAC: HDAC1, HDAC2, HDAC3, HDAC8; and Class II HDACs: HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC1O. Type I mammalian HDACs include: HDAC1, HDAC2, HDAC3, HDAC8, and HDACI1. Type II mammalian HDACs include: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC1.
As used herein, the term HDAC inhibitor refers to a compound that inhibits a histone deacetylase Class I and/or Class II enzyme. In some embodiments, the compound selectively inhibits a Class I or Class II HDAC.
By “selective” is meant at least 20%, 50%, 75%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 10-fold greater inhibition of an HDAC over another enzyme, for example a Class III or Class IV histone deacetylase. Thus, in some embodiments, the agent is selective for HDAC over a Class III histone deacetylase. In some embodiment the inhibitor is specific for a Class I or Class II and thus does not significantly inhibit HDACs of other classes.
A number of structural classes of negative regulators of HDACs (e.g., HDAC inhibitors) have been developed, for example, small molecular weight carboxylates (e.g., less than about 250 amu), hydroxamic acids, benzamides, epoxyketones, cyclic peptides, and hybrid molecules. (See, for example, Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott G K, et al. (2005) Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 45: 495-528, (including specific examples therein) which is hereby incorporated by reference in its entirety). Non-limiting examples of negative regulators of type I/II HDACs include: Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HC Toxin, Nullscript (4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA) and other short chain fatty acids), Scriptaid, Suramin Sodium, Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (i.e., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-C1-UCHA (i.e., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other inhibitors include, for example, dominant negative forms of the HDACs (e.g., catalytically inactive forms) siRNA inhibitors of the HDACs, and antibodies that specifically bind to the HDACs. Inhibitors are available, e.g., from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich. In some embodiments, VPA is a preferred histone deacetylase inhibitor.
DNA methylation is one of the most prevalent epigenetic modifications of DNA in mammalian genomes. It is achieved by DNA methyltransferases that catalyze the addition of a methyl group from S-adenosyl-L-methionine to the 5-carbon position of cytosine. Methylation at cytosine plays an important role in regulating transcription and chromatin structure. Three families of DNA methyltransferase genes have been identified in mammals. They include Dnmt1, Dnmt2 and Dnmt3. Dnmt1 is constitutively expressed in proliferating cells and its inactivation results in demethylation of genomic DNA and embryonic death. Dnmt2 is expressed at low levels in adult tissues. Its inactivation does not affect DNA methylation or maintenance of methylation. The Dnmt3 (Dnmt3a and Dnmt3b) is strongly expressed in embryonic stem cells, but is down-regulated in differentiating embryonic stem cells and in adult somatic cells.
Most mammalian transcription factors bind GC-rich DNA elements. Methylation of these elements abolishes binding. CpG methylation is shown to induce histone deacetylation, chromatin remodeling, and gene silencing through a transcription repressor complex. CpG islands are often located around the promoters of housekeeping genes and are not methylated. In contrast, the CG sequences in inactive genes are usually methylated to suppress their expression.
Examples of nucleoside DNA methyltransferase inhibitors include 5-deoxy-azacytidine (DAC), 5-azacytidine (5-aza-CR) (Vidaza), 5-aza-2′-deoxycytidine (5-aza-CdR; decitabine), 1-[beta]-D-arabinofuranosyl-5-azacytosine, dihydro-5-azacytidine, zebularine, Sinefungin (e.g., InSolution™ Sinefungin), 5-fluoro-2′-deoxycyticine (FdCyd). Examples of non-nucleoside DNA methyltransferse inhibitors (e.g., other than procaine) include: (−)-epigallocatechin-3-gallate (EGCG), RG108, hydralazine, procainamide, 1513-DMIa and 1513-DMIb which were isolated from the culture filtrate of Streptomyces sp. strain No. 1513, psammaplin, dominant negative forms of the DNA methyltransferases (e.g., catalytically inactive forms), oligonucleotides (e.g., including hairpin loops and specific antisense oligonucleotides (such as MG98)), siRNA inhibitors of the DNA methyltransferases, and antibodies that specifically bind to the DNA methyltransferases. Inhibitors are available, e.g., from Merck Biosciences.
The factor UTF1 and a p53 inactivation factor described herein can be provided in a kit.
In one embodiment, the kit includes one or two of the group consisting of UTF1 and p53 inactivation factor.
In a preferable embodiment, the kit further includes factor A selected from one or more (e.g. two, three or four) of the group consisting of Oct4, Klf4, Sox2 and c-Myc. In a more preferable embodiment, the kit further includes the compounds reported to improve reprogramming efficiency, (e.g. CHIR 99021, an HDAC inhibitor(s) such as VPA and/or a DNA methyltransferase inhibitor(s)), and, optionally (c) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of a compound(s) described herein for the methods described herein.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering the compound.
In one embodiment, the informational material can include instructions to administer the factor A selected from one or more (e.g. two, three or four) of the group consisting of Oct4, Klf4, Sox2 and c-Myc, and factor B selected from one or two of the group consisting of UTF1 and a p53 inactivation factor. In one embodiment, the informational material can include instructions to administer a compound(s) (e.g., CHIR 99021, an HDAC inhibitor(s) such as VPA and/or a DNA methyltransferase inhibitor(s)) described herein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein) (e.g., to a cell in vitro or a cell in vivo). In another embodiment, the informational material can include instructions to administer a compound(s) described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein or to a cell in vitro.
The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a compound described herein and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.
In addition to a compound(s) described herein, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a fragrance or other cosmetic ingredient, and/or an additional agent, e.g., for inducing pluripotent stem cells (e.g., in vitro) or for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than a compound described herein. In such embodiments, the kit can include instructions for admixing a compound(s) described herein and the other ingredients, or for using a compound(s) described herein together with the other ingredients, e.g., instructions on combining the two agents prior to administration.
A compound(s) described herein can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that a compound(s) described herein be substantially pure and/or sterile. When a compound(s) described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When a compound(s) described herein is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition containing a compound(s) described herein. In some embodiments, the kit contains separate containers (e.g., two separate containers for the two agents), dividers or compartments for the composition(s) and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a compound described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a compound described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In a preferred embodiment, the device is a medical implant device, e.g., packaged for surgical insertion.
As used herein, a heterologous nucleic acid, is a nucleic acid other than a native endogenous sequence for that gene. E.g., an additional copy of a gene inserted into a chromosome, or a copy on a vector, e.g., a replicative on non replicative vector which has not integrated into the chromosome.
Other features and advantages of the instant invention will become more apparent from the following examples and claims. Embodiments of the invention can include any combination of features described herein. In no case does the term “embodiment” necessarily exclude one or more other features disclosed herein, e.g., in another embodiment. The contents of all references, patent applications and patents, cited throughout this application are hereby expressly incorporated as a whole by reference.
BJ fibroblasts were purchased from ATCC (CRL-2522). hEF (human embryonic fibroblast) cells, hFSF (human fetal skin fibroblast) cells, and hAFF (human adult foreskin fibroblast) cells were purchased from the China-Japan Friendship Hospital. pOct4-GFP mice were from RIKEN BioResource Centre (Ohbo K. et. al. 2003). Above human fibroblasts, 293T cells available commercially and MEF (mouse embryonic fibroblast) cells isolated from mouse using the known method (Takahashi et. al., 2007) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, Gibco). Human embryonic stem (hES) cell lines H1, H7 and H9 were obtained from WiCell research institute (Madison, Wis.). hES cells purchased from WiCell and iPS cells prepared by the method as described herein were maintained on Mitomycin C-treated MEFs in hES Cell culture medium consisting of 80% DMEM/F12 (Invitrogen), 20% Knockout serum replacement (KSR) (Invitrogen), 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM beta-mercaptoethanol (all from Invitrogen) and 4 ng/ml basic fibroblast growth factor bFGF (P&A Biotech). Human iPS cells and hES cells were passaged by dispase II (Invitrogen). The medium was changed every day.
pLL3.7 vector (SEQ ID NO: 10) from Van Parijs L (
Lentiviral vector containing OCT4, SOX2, c-MYC, KLF4, UTF1, p53 siRNA and control p53 siRNA respectively (15 μg) together with pMDLg/pRRE, RSV/Rev and VSV-G (
To detect alkaline phosphatase (AP) activity, the cells were washed with PBS three times and stained with BCIP/NBT (Promega) for 15˜20 min (
Total RNA was isolated from cells using TRIzol (Invitrogen) and reverse-transcribed using a reverse transcription system (Promega) according to the manufacturer's protocol. PCR amplification of different genes was performed using 2× EasyTaq SuperMix (TransGen Biotech). The primers (SEQ ID NO: 21-83) used are given in Table 4 (
For embryoid body (EB) formation, iPS cells were passaged with 1 mg/ml collagenase IV (Invitrogen) and cultured in an uncoated cell culture suspension dish (Corning) in the presence of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 15% fetal bovine serum (FBS), 1 mM L-glutamine, 1% non-essential amino acids, 1% ITS, and 0.1 mM b-mercaptoethanol (all from Invitrogen). Cyst embryoid bodies were formed after 9 days of suspension culture (
2-5×106 iPS cells were injected into the kidney capsule of a SCID beige mouse (Weitongda Biotech). Teratomas were recovered 6-9 weeks after grafting. Control mice were injected with 10 million hAFF and hFSF fibroblasts and failed to form teratoma (three injections in total). Then they were embedded in paraffin and processed with hematoxylin and eosin staining and immunohistochemistry staining, the primary antibodies, monoclonal mouse anti-α-SMA (Millipore, dilution 1:200), anti-CK 19 (Dako, 1:100), anti-VIMENTIN (Dako, 1:200), anti-NEUROFILAMENT (Santa cruz, 1:200), and polyclonal rabbit anti-β-III-TUBULIN (Sigma, 1:200) were applied and incubated in a moist chamber at 4° C. overnight. Immunodetection was performed using the Anti-Mouse and the Anti-Rabbit Non-Biotin HRP Detection System (Zymed Laboratories) (
Genomic DNA was bisulfite converted by the CpGenome™ DNA modification kit (Intergen) according to the manufacturer's protocol. Modified DNA was amplified by PCR. Primer sequences are shown in Table 4. (SEQ ID NO: 84-91). PCR products were sequenced (
Total mRNA from hFSF, hAFF, hES cells (H1, H7) and the established cell lines were labeled with Cy5, hybridized to a human Oligo Microarray (Phalanx Human Whole Genome OneArray™, Phalanx Biotech) according to the manufacturer's protocol. Three technical repetitions were performed. After hybridization, arrays were scanned using GenePix 4000B scanner (Molecular Devices) and processed using the GenePix Pro 6.0 software (Molecular Devices). After removing control probes, a 14/33 presence call (SNR>=5 and foreground-background>0) was used to filter probes for the 33 microarrays, resulting in 12733 probes for further quantile normalization. The median of each sample was used for hierarchical clustering. Complete-linkage hierarchical clustering was performed using cluster 3.0 (written by Michael Eisen, Stanford University) with Spearman rank correlation coefficient as gene distances measurement and Pearson correlation coefficient as sample distances measurement (
To increase the efficiency of human iPS cell generation, we screened a panel of candidate factors (including NANOG, LIN28, DPPA4, DPPA5, ZIC3, BCL-2, h-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1 and p53-siRNA) that might facilitate the reprogramming process. The reported four human reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007) along with individual additional candidate factors, were cloned into the lentiviral vector pLL3.7-ΔU6 (
Based on the observation that AP activity was only an early event in mouse iPS cell generation (Brambrink et al., 2008; Stadtfeld et al., 2008), we further investigated whether p53 siRNA and UTF1 would enhance the efficiency of generating final stage iPS cells from human adult fibroblasts. We found that before day 18 post-transduction, the highly AP-positive colonies were mostly GFP-positive (GFP was driven by the CMV promoter in the lentiviral vector pLL3.7, and was used as an indicator of exogenous transgenes expression, FIG. 1Bh and
After day 18 post-transduction, GFP silenced colonies began to emerge (FIG. 1Bd and
We also determined whether UTF1 and p53 siRNA could substitute for the function of the reported four reprogramming factors, by withdrawing each one of them individually and using the remaining five factors to induce iPS cells from hAFF cells (6-1 strategy). We found that when OCT4, SOX2 or KLF4 were separately removed from the six factors (6-OCT4,6-SOX2, and 6-KLF4), although highly AP-positive colonies were obtained (FIG. 1Db), no iPS cell colonies were found, even extending the culture period to 60 days post-transduction. These results suggest the essential role of OCT4, SOX2 or KLF4 to fulfill the iPS cell induction process. When c-MYC was removed (6-MYC), the total colony number and highly AP-positive colony number decreased dramatically (FIG. 1Da and FIG. 1Db). However, during further cultivation, cell colonies continuously emerged and subsequently developed into iPS cell colonies. On day 35 post-transduction, the efficiency of iPS induction by 6-MYC was 71-223 iPS cell colonies from 1×105 target cells, which was comparable to that using the six factors (FIG. 1Dc). Interestingly, when c-MYC was removed (6-MYC), the colonies which emerged in further cultivation were mostly iPS colonies, compared with the large quantity of incompletely reprogrammed cell colonies when the six factors were used (
Previous reports (Nakagawa et al., 2008; Wernig et al., 2008) and our data (
We established a panel of human iPS cell lines from fibroblasts using different combinations of reprogramming factors. These cell lines were further characterized for plutipotency, gene expression profile, differentiation potential, and genomic stability (Table 1). The representative data are shown in
In order to analyze the differentiation potential of iPS cell lines, we tested their capacity for spontaneous and direct differentiation into the cell types of the three germ layers. After 8 days of floating cultivation, embryoid bodies (EB) were formed (
Recently, it is reported that iPS cells could be generated when contacting with HDAC inhibitors (and/or other compounds) by a much higher efficiency. We further tested whether UTF1 and p53 siRNA would still work in the context of HDAC inhibitors. We found that when VPA was added in the reprogramming process, UTF1 or/and p53 siRNA could still enhance the reprogramming efficiency significantly (
We also addressed whether p53 inactivation could enhance reprogramming by means other than RNA interference. A chemical compound named KU55933 (Tocris) was used, which could selectively inhibit p53 pathway. As expected, KU55933 improved reprogramming efficiency by almost 3 times when oct4, sox2 and Klf4 were introduced into mouse embryonic fibroblasts, as shown in
In summary, we have identified two factors, p53 inactivation and UTF1, that can significantly improve the reprogramming process.
| Number | Date | Country | Kind |
|---|---|---|---|
| 200810091841.2 | Apr 2008 | CN | national |
