Patent Application
20090028835
The present invention relates to the identification of a set of genes which are expressed by in vivo matured human oocytes (“transcriptome” of human oocytes) and which are involved in oogenesis, folliculogenesis, fertilization, and embryonic development. These genes and the corresponding gene products are useful for dedifferentiation or transdifferentiation of somatic cells. Additionally, these genes are useful as markers of undifferentiated cell types and for assaying whether an ESC is capable of giving rise to an oocyte and for identifying pregnancy competent oocytes.
The identification of genes and deduced pathways from the mature human oocyte will also facilitate a greater understanding of oogenesis, folliculogenesis, fertilization, and embryonic development. As described in detail infra, the present human oocyte transcriptome was derived using human metaphase II oocytes within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. In addition 101 human genes were identified as being differentially expressed by human oocytes that are potentially useful separately or in combination for effecting dedifferentiation of human somatic cells. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This invention therefore provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes will further expand the understanding of meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.
The mammalian oocyte is responsible for a number of extraordinary biological processes. It has the ability to haploidize its DNA, to reprogram sperm chromatin into a functional pronucleus, to drive early embryonic development, and to give rise to pluripotent embryonic stem cells (ESCs). Identifying the genes in the oocyte that are essential for oogenesis, folliculogenesis, fertilization, and early embryonic development will provide a valuable genomic resource in reproductive and developmental biology. However, the oocyte transcriptome and its functional significance in the human are relatively unknown because of ethical and technical limitations.
Although extensive genomic studies of oocytes and preimplantation embryos have been conducted in mouse oocytes (ref 1-6)), in human the accessibility of mature oocytes i.e., metaphase II (MII) oocytes, has been a major barrier to studying oocyte genomics using microarrays. Attempts have been made to address this problem by using candidate gene approaches employing RT-PCR and differential display (refs 7-22). In addition, serial analysis of gene expression (SAGE) and cDNA libraries was generated from human oocytes, and SAGE tags and expressed sequence tags were sequenced for rapid gene discovery and expression profiling in the oocytes (see reviews in refs. 23 and 24). However, these molecular approaches resulted in a small number of genes analyzed in each sample. Recently, four reports described initial transcriptome analyses of human oocytes using microarrays (refs. 25-28). Although they provided valuable information, these studies did not present a comprehensive picture of the human oocyte transcriptome because of a number of biological and technical constraints. Among the biological impediments are the use of discarded human oocytes that have failed to fertilize (refs 25, 28), limited coverage of the microarrays (refs. 25, 27), in some cases lack of sufficient biological replications (refs 26,28), and technical issues (ref. 27). Among the technical shortcomings, the most important is the use of a potentially unfaithful RNA amplification protocol. Li et al. (ref. 27) seem to have synthesized the first-strand cDNA using only a simple oligo(dT) primer, which makes target amplification unfeasible (ref 27); however, the actual procedure used for RNA amplification is unclear. Although we recognize this issue could have been a mistake on their described-published protocol, the actual procedure used for RNA amplification remains elusive.
The shortcomings of the prior art are disadvantageous as the identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development.
This invention relates to a novel transcriptome or set of genes which are differentially expressed and/or upregulated by human oocytes.
This invention also relates to the identification of a discrete set of genes which are uipregulated by in vivo matured human oocytes and the use of these genes and the corresponding gene products for the dedifferentiation and transdifferentiation of somatic cells particular somatic cells derived from a subject that is to receive transplanted cells for treatment of a particular condition such as cancer or autoimmunity. The use of these genes and/or gene products produced using such donor transdifferentiated or dedifferentiated cells will allow for the production of desired immature and/or somatic cell types that are compatible for a desired donor and which therefore are suitable for human cell and gene therapy without the need for human nuclear transfer (“human therapeutic cloning”) which has ethical concerns because such methods may result in the destruction of human embryos.
In addition, the present invention relates to the use of these genes and the corresponding gene products as markers of dedifferentiated cells such as human adult embryonic stem cells. Because these genes are expressed by in vivo matured human oocytes, it is anticipated that some of these genes will be expressed by other human immature cell types such as adult stem cells and cancer stem line cells.
Also, the invention relates to the use of these genes and gene products and probes specific thereto such as antibodies and oligonucleotides complementary thereto for the isolation and enrichment of such stem cells from heterogeneous cell samples such as by FACS, magnetic bead cell separations, and other cell separation methods.
Also, the present invention relates to the production of mammalian oocytes wherein one or more of these genes or an ortholog thereof in the case of non-human oocytes are “knocked out” or expressed under regulatable conditions in order to study the effect of these genes on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, morphogenesis, oogenesis, folliculogenesis, and embryonic development.
Further, the invention relates to dedifferentiated or transdifferentiated somatic cells produced using one or more of the genes and gene products disclosed herein that are upregulated on in vivo matured human oocytes.
In addition, the invention provides novel and improved cell and gene therapies using the transdifferentiated and dedifferentiated somatic cells produced by introduction of one or more of the disclosed genes and gene products comprised in the human oocyte transcriptome which is disclosed herein. In addition, the invention relates to culture medium containing one or more of these gene products corresponding to the disclosed human oocyte transcriptome or their non-human orthologs and variants and the use thereof for dedifferentiation and/or transdifferentiation of desired somatic or embryonic cell types.
Also, the invention relates to the use of these genes in order to establish a signature of normal human oocytes, i.e., human oocytes that are pregnancy competent and which when fertilized are capable of giving rise to a normal pregnancy.
Prior to discussing the invention in more detail, the following definitions are provided. Otherwise all words and phrases in this application are to be construed by their ordinary meaning, as they would be interpreted by an ordinary skilled artisan within the context of the invention.
“Transcriptome” refers to a set of genes expressed by a specific cell such as an oocyte or a somatic cell type.
“Human Oocyte Transcriptome” herein refers to a set of genes upregulated by human oocytes that include genes that encode polypeptides that induce the transdifferentiation or dedifferentiation of somatic cells, preferably human somatic cells from a donor or recipient that is to undergo cell therapy.
“Pluripotent cell” refers to a cell that is capable of giving rise to all 3 cell lineages, i.e., ectoderm, endoderm and mesoderm cells.
“Multipotent” refers to a cell that is capable of giving rise to more than 1 cell lineage.
“Totipotent cell” is an undifferentiated cell such as embryonic cell such as an oocyte that is capable of giving rise to a viable offspring under appropriate conditions.
“Embryonic Stem Cell or ESC” is a cell that is capable of giving rise to all 3 lineages. ESCs may be derived from early stage embryos, umbilical cord and other embryo tissue material as well as from nuclear transfer derived embryos.
“Adult stem cell” is a cell capable of giving rise to different somatic cells of a specific lineage, e.g., immune stem cells, hematopoietic stem cells, neural stem cells, pancreatic stem cells and the like which cells are present in very few numbers in adult tissues and which cells unlike other adult somatic cells may be isolated and induced to differentiate resulting in the production of specific somatic cell lineages such as neural cells if the adult stem cell is a neural stem cell.
“Pregnancy-competent oocytes”: refers to a female gamete or egg that when fertilized by natural or artificial means is capable of yielding a viable pregnancy when it is comprised in a suitable uterine environment.
“Viable-pregnancy”: refers to the development of a fertilized oocyte when contained in a suitable uterine environment and its development into a viable fetus, which in turn develops into a viable offspring absent a procedure or event that terminates said pregnancy. “Cumulus cell” refers to a cell comprised in a mass of cells that surrounds an oocyte. These cells are believed to be involved in providing an oocyte nutritional and or other requirements that are necessary to yield an oocyte which upon fertilization is “pregnancy competent”.
“Differential gene expression” refer to genes the expression of which varies within a tissue of interest; herein preferably an oocyte.
“Real Time RT-PCR”: refers to a method or device used therein that allows for the simultaneous amplification and quantification of specific RNA transcripts in a sample.
“SAGE” is an acronym for “Serial Analysis of Gene Expression”. “Microarray analysis”: refers to the quantification of the expression levels of specific genes in a particular sample, e.g., tissue or cell sample.
“Pregnancy signature”: refers to a phrase coined by the inventors which refers to the characteristics levels of expression of a set of one or more genes, preferably at least 5, more preferably at least 10 to 20 genes, and still more preferably, at least 50 to 100 genes, that are expressed at characteristic levels in oocytes or oocyte associated cells, preferably cumulus cells, that surround “pregnancy competent” oocytes. This is intended to encompass the level at which the gene is expressed and the distribution of gene expression within cells analyzed.
“Pregnancy signature gene”: refers to a gene which is expressed at characteristic levels by a cell, e.g., cumulus cell, on a “pregnancy competent” oocyte.
“IVF”: refers to in vitro fertilization. “Zona pellucida” refers to the outermost region of an oocyte.
“Method for detecting differential expressed genes” encompasses any known method for evaluating differential gene expression. Examples include indexing differential display reverse transcription polymerase chain reaction (DDRT-PCR); subtractive mRNA hybridization, the use of nucleic acid arrays or microarrays; SAGE (Serial Analysis of Gene Expression. (SAGE) and real time PCR(RT-PCR). For example, differential levels of a transcribed gene in an oocyte cell can be detected by use of Northern blotting, and/or RT-PCR. CRL amplification protocol refers to the novel total RNA amplification protocol that combines template-switching PCR and T7 based amplification methods. This protocol is well suited for samples wherein only a few cells or limited total RNA is available.
“EASE” is a gene ontology protocol that from a list of genes forms subgroups based on functional categories assigned to each gene based on the probability of seeing the number of subgroup genes within a category given the frequency of genes from that category appearing on the microarray.
“Dedifferentiated or transdifferentiated or reprogrammed somatic cell” refers to a somatic cell which is converted into a less mature cell, e.g., a stem or stem-like cell or a rejuvenated cell that has a longer lifespan than the parent somatic cell or is converted into a different somatic cell lineage. This is effected by incorporating into the cell one or more of the 101 genes disclosed herein that are differentially expressed by mature human oocytes, preferably expressed under the control of a regulatable promoter, or by contacting the cell or the nucleus or chromatin mass derived therefrom with a medium containing at least one gene product encoded by the 101 genes disclosed herein or a non-human ortholog thereof. In these methods the somatic cell, e.g., from a donor with a disease treatable by cell therapy is introduced (e.g., via electroporation, injection, infection) one or more human genes or a vector(s) containing, wherein said gene or genes are expressed by a mature human oocyte or said somatic cell or the nucleus thereof is cultured in a medium containing one or more gene products expressed by mature human oocytes that results in said somatic cell converting into a less differentiated cell, e.g., a stem cell or into a cell with an increased lifespan as evidenced by an increased telomere length or increased cell doublings or which somatic cell converts into a different somatic cell type. Dedifferentiation can be detected by screening for markers characteristic of pluripotent cell types, altered telomere length, increased number of cell doublings until senescence or by detecting for telomerase which is only expressed by pluripotent or immortal cells such as ESCs and cancer cells.
“Nuclear transfer embryo” refers to an embryo created by fusing or inserting a somatic cell or the nucleus or chromosomes thereof with an oocyte or other embryonic cell that is enucleated before, during or after fusion or insertion.
“Parthenogenic embryo” refers to an embryo that is produced using only male or female gametes. Typically, these parthenogenic embryos are incapable of giving rise to viable offspring.
“Gene Contained in Human Oocyte Transcriptome or an ortholog or variant thereof” refers to one or more of the human oocyte expressed genes disclosed herein, i.e., the genes contained in
This invention provides a set of 101 genes which are differentially expressed and/or upregulated by normal mature human oocytes (transcriptome). These genes and the sequences which are contained in
Particularly, these genes were identified using human metaphase II oocytes assayed within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol disclosed herein. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This invention therefore provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes.
In addition, the present transcriptome was identified using young oocytes, as opposed to aged and fertilized ones, which could have quite significantly different expression profiles. Also, the inventors used the most comprehensive human microarray platform; and took advantage of publicly available gene expression databases to interpret our own data in a more meaningful way. Thus, the present invention was conducted using materials and methods that were designed to identify the gene transcripts present in young, untreated MII oocytes within minutes after isolation from the ovary in three independent replicates and to compare these genes with a reference RNA (a mixture of total RNA from 10 different normal human tissues not including the ovary) by using Affymetrix GeneChip technology.
More specifically, to achieve this goal, a protocol that combined template-switching PCR and T7-based amplification methods was developed for the analysis of gene expression in samples of small quantity. The inventors amplified RNA from the oocyte and reference samples. Results were later compared with available transcriptome databases of mouse oocytes, and human ESCs (hESCs) and mouse ESCs (mESCs).
Using these methods, the inventors provide herein the transcript profile of in vivo matured human MII oocytes using the most recent Affymetrix human GeneChip array, interrogating >47,000 transcripts including 38,500 well characterized human genes.
This invention particularly provides a transcriptome of 101 genes expressed by human oocytes. These genes and the orthologs of these genes are involved in the differentiation based on their specific expression in oocytes. Therefore, the introduction of one or more of these genes or the corresponding gene products should result in the transdifferentiation or dedifferentiation of a desired somatic cell into another cell type and/or lengthen the lifespan of said cell.
Particularly the introduction of at least one of said genes or gene products will result in the partial or complete dedifferentiation of a desired somatic cell into a pluripotent or multipotent cell., e.g., an adult stem cell or an embryonic cell type. Cells which transdifferentiate or dedifferentiate may be detected by screening by at least one marker that is specific for the particular cell type. For example, it is known that embryonic stem cells express certain genes such as Oct4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase. Also, dedifferentiated cells or cells having enhanced lifespan may be detected based on an analysis of telomerase expression or telomere length. Cells having longer telomeres relative to stating somatic cells correlate to an enhanced cell lifespan.
These transdifferentiated cells may be used e.g., for cell therapy or for study of the differentiation process. Diseases treatable by cell therapy include by way of example cancer, autoimmunity, allergy, inflammatory conditions, infection. Cancers treatable by use of cell therapy include solid and non-solid tumor associated cancers and include by way of example hematological cancers such as myeloma, lymphoma, leukemia; sarcomas, melanomas, lung cancers, pancreatic, neurological cancers such as neuroblastomas, stomach, colon, liver, gall bladder, esophageal, tracheal, head and neck, cancers of the tongue and lip, ovarian, breast, cervical, prostate, testicular, bone and other cancers. In addition cell therapy is useful in alleviating the effects of specific treatments such as radiation and chemotherapy which may deplete specific cells such as bone marrow. Further the subject cell therapy may be used for treating infectious disease such as viral or bacterial or parasite associated diseases such as HIV. Also the subject cell therapy may be used in treating autoimmune conditions wherein the host autoimmune reaction may result in killing or depletion of host cells such as immune cells or other essential cell.
Reprogrammed or dedifferentiated or transdifferentiated cells generated from these methods may be used to replace cells in a mammal in need of a particular cell type. These methods may be used to either directly produce cells of the desired cell type or to produce undifferentiated cells which may be subsequently differentiated into the desired cell type. For example, stem cells may be differentiated in vitro by culturing them under the appropriate conditions or differentiated in vivo after administration to an appropriate region in a mammal. To optimize phenotypic and functional changes, reprogrammed cells can be transplanted into the organ (e.g., a heart) where they are intended to function in an animal model or in human patients shortly after dedifferentiation or transdifferentiation (e.g., after 1, 2, 3, 5, 7, or more days). The resultant cells implanted in an organ may be reprogrammed to a greater extent than cells grown in culture prior to transplantation. Cells implanted in an animal organ may be removed from the organ and transplanted into a recipient mammal such as a human, or the animal organ may be transplanted into the recipient.
To increase the length of time the cell, nuclei, or chromatin mass may be reprogrammed in vitro prior to administration to a mammal for the treatment of disease, the donor cell may be optionally modified by the transient transfection of a plasmid containing an oncogene flanked by loxP sites for the Cre recombinase and containing a nucleic acid encoding the Cre recombinase under the control of an inducible promoter (Cheng et al., Nucleic Acids Res. 28(24):E108, 2000). The insertion of this plasmid results in the controlled immortalization of the cell. After the cell is reprogrammed into the desired cell-type and is ready to be administered to a mammal, the loxP-oncogene-loxP cassette may be removed from the plasmid by the induction of the Cre recombinase which causes site-specific recombination and loss of the cassette from the plasmid. Due to the removal of the cassette containing the oncogene, the cell is no longer immortalized and may be administered to the mammal without causing the formation of a cancerous tumor.
Examples of medical applications for these cells include the administration of neuronal cells to an appropriate area in the human nervous system to treat, prevent, or stabilize a neurological disease such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or ALS; or a spinal cord injury. In particular, degenerating or injured neuronal cells may be replaced by the corresponding cells from a mammal. This transplantation method may also be used to treat, prevent, or stabilize autoimmune diseases including, but not limited to, insulin dependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris, multiple sclerosis, and myasthenia gravis. In these procedures, the cells that are attacked by the recipient's own immune system may be replaced by transplanted cells. In particular, insulin-producing cells may be administered to the mammal for the treatment or prevention of diabetes, or oligodendroglial precursor cells may be transplanted for the treatment or prevention of multiple sclerosis. For the treatment or prevention of endocrine conditions, reprogrammed cells that produce a hormone, such as a growth factor, thyroid hormone, thyroid-stimulating hormone, parathyroid hormone, steroid, serotonin, epinephrine, or norepinephrine may be administered to a mammal. Additionally, reprogrammed epithelial cells may be administered to repair damage to the lining of a body cavity or organ, such as a lung, gut, exocrine gland, or urogenital tract. It is also contemplated that reprogrammed cells may be administered to a mammal to treat damage or deficiency of cells in an organ, muscle, or other body structure or to form an organ, muscle, or other body structure. Desirable organs include the bladder, brain, nervous tissue, esophagus, fallopian tube, heart, pancreas, intestines, gallbladder, kidney, liver, lung, ovaries, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, and uterus. Also, these cells may also be combined with a matrix to form a tissue or organ in vitro or in vivo that may be used to repair or replace a tissue or organ in a recipient mammal. For example, reprogrammed cells may be cultured in vitro in the presence of a matrix to produce a tissue or organ of the urogenital system, such as the bladder, clitoris, corpus cavermosum, kidney, testis, ureter, uretal valve, or urethra, which may then be transplanted into a mammal (Atala, Curr. Opin. Urol. 9(6):517-526, 1999). In another transplant application, synthetic blood vessels are formed in vitro by culturing reprogrammed cells in the presence of an appropriate matrix, and then the vessels are transplanted into a mammal for the treatment or prevention of a cardiovascular or circulatory condition. For the generation of donor cartilage or bone tissue, reprogrammed cells such as chondrocytes or osteocytes are cultured in vitro in the presence of a matrix under conditions that allow the formation of cartilage or bone, and then the matrix containing the donor tissue is administered to a mammal. Alternatively, a mixture of the cells and a matrix may be administered to a mammal for the formation of the desired tissue in vivo. Preferably, the cells are attached to the surface of the matrix or encapsulated by the matrix. Examples of matrices that may be used for the formation of donor tissues or organs include collagen matrices, carbon fibers, polyvinyl alcohol sponges, acrylateamide sponges, fibrin-thrombin gels, hyaluronic acid-based polymers, and synthetic polymer matrices containing polyanhydride, polyorthoester, polyglycolic acid, or a combination thereof (see, for example, U.S. Pat. Nos. 4,846,835; 4,642,120; 5,786,217; and 5,041,138).
Additionally, these dedifferentiated somatic cells may be used to produce artificial tissues and organs by culturing said dedifferentiated cells in vitro e.g., in cell culture apparatus that are designed to facilitate the formation of desired cell structure and morphology. Additionally, these cells may be introduced into non-human animals as xenografted cells for example by injecting the dedifferentiated into desired organs. For example, dedifferentiated cells may be used to study the effect of dedifferentiated cardiac cells on damaged heart tissue to determine whether these cells promote the healing or regeneration process. Alternatively, dedifferentiated immune cells may be introduced into immunodeficient animals to assess whether this results in restoration of immune function.
In addition probes specific to the subject human genes or gene products may be used to identify dedifferentiated cells in a mixed cell population. For example, these probes such as labeled antibodies or oligos specific to the subject human genes or gene products differentially expressed by mature human oocytes may be used to identify and/or isolate adult stem cells contained in adipocyte tissue (fat) or other tissue that may contain adult stem cells.
Also, these probes may be used to assess the viability and/or pregnancy competency of oocytes from donors that may have oocyte related pregnancy problems e.g., as a result of disease, genetics, age, or environmental insult. Since the subject transcriptome was derived from oocytes from young apparently healthy donors the expressed genes should include those which are required for the oocyte to be pregnancy competent upon natural or in vitro fertilization.
In order to describe the invention in greater detail the following experimental examples and results are provided below.
The Materials and Methods below were used to derive the human transcriptome or set of genes upregulated by in vivo matured metaphase II human oocytes.
Oocyte Collection Total RNA Extraction, and Reference RNA.
Human oocytes were obtained from three patients undergoing an assisted reproductive treatment (ART) at the Unit of Reproductive Medicine of Clinica Las Condes, Santiago, Chile. It is important to emphasize that the routine in vitro fertilization protocol at Clinica Las Condes calls for fertilizing only those oocytes that will be transferred into the uterus of the patient. Therefore, there is always a surplus of oocytes. We then had the opportunity to use specific criteria to select donors as follows: (i) <35 years old, (ii) reproductively healthy with regular ovulatory cycles, (iii) male factor as the only cause of infertility, and (iv) considerable number of developing follicles that assured spared oocytes. The experimental protocol was reviewed and approved by a local independent Ethics Review Board. All donors signed informed consent. At the time this manuscript was submitted, all three donors had already conceived; two of them got pregnant during the ART cycle in which our samples were collected, and the third one got pregnant after a spontaneous cycle with artificial insemination using donated sperm. Ovarian stimulation, oocyte retrieval, and cell lysis were performed as described in Supporting Materials and Methods, which is published as supporting information (See Ref. 50).
Three groups of 10 oocytes each were used. Total RNA was isolated following the guanidium thiocyanate method (Ref. 45) by using the PicoPure RNA isolation kit (Arcturus, Sunnyvale, Calif.) following the manufacturer's instructions. However, only 6.5 μl of elution buffer (Arcturus) was used, and the elution was repeated at least three times by using the first eluate. All RNA samples within the purification column were treated with the RNase-Free DNase (Qiagen, Valencia, Calif.). Extracted RNA was stored at −80° C. until used as template for cDNA synthesis. The quality and quantity of extracted total RNA from 8 matured oocytes (independent from the 30 oocytes used in this study) was evaluated on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.). Each mature oocyte was found to have ≈330 pg of total RNA when the Arcturus RNA isolation kit was used. The quality of RNA was intact as shown in
First Strand cDNA Synthesis and cDNA Purification
The following reagents were added to each 0.5 ml of RNase-free tube: 5 μl total RNA (i.e., 3 ng for the reference and 3 ng for the oocyte samples) and 300 ng of an anchored T7-Oligo(dT)24V promoter primer (Ambion). The reaction tubes were incubated in a preheated PCR machine at 70° C. for 2 min and transferred to ice. After denaturation, the following reagents were added to each reaction tube: 1.4 μl of SMART II A oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTACGCGrGrGr-3′) (Clontech), 4 μl of 5× first-strand buffer, 2 μl of 20 mM DTT, 0.6 μl of 5 mg/ml T4 Gene 32 Protein (Roche, Indianapolis, Ind.), 2 μl of 10 mM dNTPs, 20 units of RNase inhibitor (Ambion), and 1 μl of PowerScript Reverse Transcriptase (Clontech). The final first-strand reaction volume was 20 μl for all experiments. After gently mixing, reaction tubes were incubated at 42° C. for 60 min in a hot-lid thermal cycler. The reaction was terminated by heating at 70° C. for 15 min and purified by NucleoSpin Extraction Kit (Clontech) following the manufacturer's instructions.
Double Stranded cDNA Synthesis by L:omg-Distance PCR and cDNA Purification PCR
Advantage 2 mix (9 μl) was prepared as follows: 5 μl of 10×PCR Advantage buffer (Clontech), 1 μl of 10 mM dNTPs, 100 ng of 5′ SMART upper primer (5′-AAGCAGTGGTATCAACGCAGAGTA-3′), 100 ng of 3′ SMART lower primer (5′-CGGTAATACGACTCACTATAGGGAGAA-3′), and 1 μl of Polymerase Mix Advantage 2 (Clontech). This mix was added to 41 μl of the first-strand cDNA synthesis reaction product, and thermal cycling was carried out in the following conditions: 95° C. for 1 min, followed by 15 cycles, each consisting of denaturation at 94° C. for 30 s, annealing at 62° C. for 30 s, and extension at 68° C. for 10 min. The cDNA was purified by the NucleoSpin Extraction Kit (Clontech).
For in vitro transcription, biotin-labeled aRNA purification and aRNA fragmentation, hybridization, washing, staining and imaging, and RT-PCR analysis, see Supporting Materials and Methods.
Transcriptional profile of each sample was probed by using Affymetrix Human Genome U133 Plus 2.0 GeneChips. The raw data obtained after scanning the arrays were analyzed by dChip (ref. 46). A smoothing spline normalization method was applied before obtaining model-based gene expression indices, also known as signal values. There were no outliers identified by dChip so all samples were carried on for subsequent analysis.
When comparing two groups of samples to identify genes enriched in a given group, we used the lower confidence bound (LCB) of the fold change (FC) between the two groups as the cut-off criteria. If 90% LCB of FC between the two groups was >2, the corresponding gene was considered to be differentially expressed (DE). LCB is a stringent estimate of the FC and has been shown to be the better ranking statistic (Ref 46). Recently, dChip's LCB method for assessing DE genes has been shown to be superior to other commonly used approaches, such as MAS 5.0 and Robust Multiarray Average-based methods (Refs. 47 and 48).
Pathways analysis was performed by using Ingenuity Software Knowledge Base (Redwood City, Calif.), which is a manually created database of previously published findings on mammalian biology from the public literature. We used the network analysis, using the knowledge base to identify interactions of input genes within the context of known biological pathways.
Gene Ontology was performed by using the EASE software package). Given a list of genes, EASE forms subgroups based on the functional categories assigned to each gene. EASE assigns a significance level (EASE score) to the functional category based on the probability of seeing the number of subgroup genes within a category given the frequency of genes from that category appearing on the microarray (Ref 29).
Comparison with External Data Sets.
Mouse MII oocyte transcriptome data were obtained from Su et al. (Ref 33), who used custom designed Affymetrix chips to obtain gene expression profiles of oocytes and 60 other mouse tissue types. Using their expression database, we identified 3,617 differentially up-regulated transcripts in the mouse oocyte using the median expression value of the remaining 60 samples as the baseline (see Data Set 8, which is available in Ref 50. We selected transcripts with an expression value in oocyte samples that are 2-fold higher than the baseline.
Human ESC data were derived from the work of Sato et al. (Ref 44), who profiled human stem cells and their differentiated counterparts using Affymetrix HG-U133A representing z22,000 transcripts.
We analyzed the raw data using dChip and identified 1,626 hESC genes by selecting transcripts significantly up-regulated in human stem cells compared with their differentiated counterparts (see Data Set 9, which is available in Ref 50).
Finally, for mESCs, we used a list of 1,687 differentially up-regulated mESC genes published by Fortunel et al. (49), which were identified by comparing mESCs to differentiated cells by using Affymetrix MG-U74Av2 chips representing 12,000 transcripts (see Data Set 10, which is available in Ref 50). We used commercially available Affymetrix's NetAffx tool) for mapping genes across organisms and platforms used in the respective studies.
Ovarian stimulation was performed under Gn-RH analog suppression [leuprolide acetate (Lupron); Abbott, Abbott Park, Ill.] in a daily s.c. dose of 0.5 mg. Recombinant FSH (rFSH, Gonal-f-Serono or Puregon; Organon, Roseland, N.J.) was administered in daily doses that ranged between 200 and 300 units, starting the second day of the mense. Follicular growth and estradiol levels were monitored every 2 to 3 days until follicles had a mean diameter between 18 and 20 mm. Oocyte maturation was achieved by an injection of 10,000 units of hCG (Pregnyl; Organon). Oocytes were retrieved from the ovary by aspiration using guided transvaginal ultrasound 36 h after hCG administration. Three hours after retrieval, oocytes were denuded from surrounding corona and cumulus cells by a brief incubation (10-30 s) in 80 units/ml hyaluronidase solution (LifeGlobal, Guilford, Conn.) and subsequent pipetting to completely eliminate other cells. Oocytes were then observed at high magnification to confirm maturity (metaphase II stage) and to confirm the absence of other cells. Each oocyte was rinsed in sterile PBS and lysed in 100 μl of extraction buffer (XB, Arcturus, Mountain View, Calif.) in an RNase/DNase/Pyrogen-free 0.5-ml microcentrifuge tube. Each sample was incubated for 30 min at 42° C., centrifuged at 3,000×g for 2 min, and stored in liquid nitrogen until use.
Transcription (IVT), Biotin-Labeled Amplified RNA (aRNA) Purification, and aRNA Fragmentation.
The purified double-stranded cDNA containing the T7 promoter sequence was used as a template for IVT-labeling assays in the presence of biotin-labeled ribonucleotides, using the BioArray HighYield RNA Transcript Labeling kit with T7 RNA polymerase (ENZO, Farmingdale, N.Y.) as described by the manufacturer. The biotin-labeled aRNA was purified using RNeasy mini columns (RNeasy Mini Kit; Qiagen, Valencia, Calif.). In vitro transcription of the cDNA for each replicate yielded 70-90 μg of biotinylated aRNA, and 15 μg of the labeled aRNA was fragmented at 94° C. for 35 min in 1× fragmentation buffer (40 mM Tris-acetate, pH 8.1/100 mM KOAc/30 mM MgOAc).
The Affymetrix GeneChip system was used for hybridization, staining, and imaging of the arrays. Hybridization cocktails of 300 μl containing 15 μg of fragmented biotin-labeled aRNA and biotinylated exogenous hybridization controls (50 pM control Oligo B2, Eukaryotic hybridization controls) (BioB at 1.5 pM, BioC at 5 pM, BioD at 25 pM, and CreX at 100 pM), herring sperm DNA (0.1 mg/ml), BSA (0.5 mg/ml) in buffer (100 mM Mes/1M NaCl/20 mM EDTA/0.01% Tween 20) were hybridized to the GeneChip Human Genome U133 plus 2.0 array (Affymetrix, Santa Clara, Calif.). Hybridizations were performed automatically, and each array was prehybridized with all components except the fragmented biotin-labeled aRNA in a chamber at 45° C. for 15 min with rotation at 60 rpm. The prehybridized array was then hybridized with the aRNA mixture for 16 h under the prehybridization conditions. After hybridization, the mixture was removed from chip, and the array was filled with nonstringent wash buffer (6×SSPE and 0.01% Tween 20). The arrays were washed according to Affymetrix protocol on a Fluidics station using nonstringent and stringent (100 mM Mes/0.1 M NaCl/0.01% Tween 20) wash buffers. For the detection of hybridized fragments, the array was stained using SAPE (streptavidin-linked to phycoerythrin) stain and antibody solutions. SAPE stain solution (600 μl) contained 2 mg/ml BSA, 10 μg/ml streptavidin phycoerythrin (SAPE) in 100 mM Mes, 1M NaCl, and 0.05% Tween 20. The antibody solution (600 μl) also contained 2 mg/ml BSA, 0.1 mg/ml goat IgG, 3 μg/ml biotinylated anti-streptavidin antibodies in 100 mM Mes, 1M NaCl, and 0.05% Tween 20. The order of staining is SAPE, antibody, and second SAPE.
The arrays were scanned using Affymetrix's high density GeneArray Scanner 3000 and imaged using Affymetrix GeneChip Operating Software (GCOS). The GCOS expression data report was generated for each sample and was used to judge the quality of sample preparation and hybridization. The report included information about noise, background, and percentage of probe sets called present based on the manufacturer threshold and software settings. Information about performance of exogenously added prokaryotic hybridization control genes such as BioB, BioC, and BioD of the Escherichia coli biotin synthesis pathway and the ratio of intensities of 3′ probes to 5′ probes for housekeeping genes such as GAPDH and β-actin were also included in the expression report file (for independent oocyte and reference samples, see Expression Report Files 1-6, which are available at www.canr.msu.edu/dept/ans/community/people/cibelli_jose.html).
Equal amounts of the remaining long-distance PCR reactions diluted 1:1 with sterile H2O were amplified by gene-specific primers (Table 4, which is published as supporting information on the PNAS web site). After an initial incubation at 94° C. for 3 min, the RT-PCR was carried out at 94° C. for 45 s, 60° C. for 30 s, 72° C. for 40 s for 30 cycles. Upon the completion of PCR, the reaction was incubated at 72° C. for an additional 10 min. RT-PCR products were electrophoresed on a 1.5% agarose gel and documented with a Gel Documentation System (Bio-Rad, Hercules, Calif.).
A critical step in the analysis of gene expression on small samples is the faithful amplification of mRNA molecules present in the sample. We have designed a PCR-based amplification system using the combination of SMART II A oligonucleotide (Clontech, Mountain View, Calif.) and T7-Oligo(dT) promoter primers (CRL RNA amplification protocol) (
A selected list of genes was used to validate the microarray results by RT-PCR (
We generated a database of the human oocyte transcriptome by comparing the transcripts in the oocyte with the reference samples, which contain mRNA from several somatic tissues. A complete list of up- and down-regulated genes and functional, comparative, and correlation analyses are available (see Data Sets 1 and 2 in Ref. 50. In addition although the oocytes were thoroughly denuded manually from their surrounding cells, we were concerned over the risk for potential contamination of RNA belonging to cumulus cells. We then specifically checked for the levels of expression of cumulus cell-specific genes, such as GREMI, PTGS2 and PTX3, and found them absent or down-regulated in the oocyte RNA samples.
Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Genes up-regulated in oocyte samples included most of the well-known germ cell-specific genes, such as FIGLA, STELLA, VASA, DAZL, GDF9, ZP1, ZP2, MOS, OCT4, NPM2, and H1FOO. Using Ingenuity Software Knowledge Base (Redwood City, Calif.), we confirmed the presence of pathways previously described in the mouse, in particular the TGF-β pathway (See
To examine the biological processes performed by the oocyte, we implemented Expression Analysis Systematic Explorer (EASE) (Ref 29), contrasting the genes overexpressed in the oocyte with all of the genes present in the Affymetrix chip (Table 1, which is published as supporting information on the PNAS web site). One of the top overrepresented categories found in oocytes was related to RNA metabolism. This finding is in agreement with the fact that oocytes store RNA to support the events of fertilization and early embryonic development until the embryonic genome is activated (Ref 30, 31)). DNA metabolism and chromatin modification were also overrepresented categories, in agreement with the need of the oocyte to remodel the sperm chromatin upon fertilization. Detection of gametogenesis and reproduction as overrepresented categories further suggests the accuracy of this transcriptional profiling. An important category highly represented in the oocyte was related to nucleic acid metabolism and regulation of transcription. Although transcriptionally silent at the MII stage, the oocyte is very active in transcription and translation throughout its growth phase and must be prepared to initiate transcription during embryonic genome activation at the four- to eight-cell stage in human (32). Many of the genes in this category represent Zinc-finger proteins that are not yet fully characterized, providing an opportunity to discover new transcriptional regulatory networks that operate during embryonic genome activation.
Chromatin remodeling genes are also represented in the human oocyte. Genes in this category included the following: DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), histone acetyltransferases (NCOA1 and -3, SRCAP, GCN5L2, and TADA2L), histone deacetylases (HDAC3, HDAC9, and SIRT7), methyl-CpG-binding proteins (MBD2 and MBD4), histone methyltransferases (EHMT1 and SET8), ATP-dependent remodeling complexes (SMARCA1, SMARCA5, SMARCAD1, SMARCC2, and SMARCD1), and other chromatin-modifying genes (ESR1, NCOA6, HMGB3, HMGN1, and HMGA1). These Gene Ontology results not only validate our transcriptome analysis when compared with candidate gene analysis already reported in other species but more importantly, shed new light into a large number of biological processes that take place in the human oocyte.
Mouse has been the best model for genetic studies, and several groups have already reported the transcriptome analysis of mouse oocytes (Ref 4, 6). In an effort to find differences and similarities between the human and mouse oocyte, we compared our human oocyte transcriptome results with that of mouse oocyte transcriptome derived from data of Su et al. (33). We intersected differentially up-regulated genes in the human and the mouse oocyte transcriptome and found a set of 1,587 genes to be in common, indicating genes of conserved function in mammalian oocytes (
The oocyte is derived from germ cell precursors believed to have segregated from pluripotent precursors before somatic tissue differentiation (Ref 37). Primordial germ cells (PGCs) undergo mitotic proliferation followed by meiosis. By the time the oocyte reaches the MII stage, it is already a highly specialized cell capable of remodeling the sperm nucleus and restoring totipotency to the diploid zygote. In addition, somatic cell nuclear transfer (SCNT) into enucleated oocytes has shown that, when challenged with a somatic nucleus, the oocyte cytosol will attempt to completely erase the somatic epigenetic phenotype and transform the nucleus to a totipotent state. Although failures in this epigenetic reprogramming have been reported elsewhere, there are reported cases in which animals produced by SCNT have developed normally (Ref 38). Reinforcing the notion that, when SCNT is performed under ideal circumstances (yet to be described), the oocyte cytosol can turn a somatic nucleus into a totipotent one. Recent somatic cell-ESC fusion experiments suggest that ESCs retain similar as yet undefined components that can initiate the reprogramming of introduced somatic nucleus to confer pluripotency to the somatic nuclei (as measured by phenotypic and by transcriptional analyses). In this respect, the cytoplasmic environment of both ESCs and oocytes shares the capacity to reprogram a somatic nucleus (Ref 39-41). Furthermore, recent work suggests that mESCs can give rise to PGCs that can differentiate into cells similar to oocytes and sperm in a period significantly shorter when compared with in vivo gametogenesis (Ref 42,43). Taken together, this evidence indicates that there is a common set of genes between oocytes and ESCs responsible for reprogramming somatic cells. To identify these genes, differentially up-regulated transcripts in the oocyte were compared and intersected with recently published data for genes that are expressed preferentially in ESCs (Ref 44). Our analysis of the Sato et al. (Ref 44) data revealed 1,626 hESC differentially up-regulated genes. When these hESC genes were intersected with our human differentially up-regulated oocyte transcripts, we found an overlap of 388 transcripts (See
Results
cerevisiae)
pombe)
5′-CTCAAATCTCCTCCGAGACG-3′
5′-TGAAGTGGCTTGGTGTCTTG-3′
5′-GGCATCCTCACCCTGAAGTA-3′
5′-CCATCTCTTGCTCGAAGTCC-3′
The Tables referenced in the Examples are set forth supra. In addition
This invention provides a comprehensive expression baseline of gene transcripts present in in vivo matured human MII oocytes. Using the most recent Affymetrix Human GeneChip, we have identified 5,331 transcripts highly expressed in human oocytes, including well known genes such as FIGLA, STELLA, VASA, DAZL, GDF9, ZP1, ZP2, MOS, OCT4, NPM2, NALP5/MATER, ZAR1, and H1FOO. More importantly, 1,430 of these up-regulated genes have unknown functions, arguing for the need for further studies aimed to elucidate the functional role of these genes in the human oocyte.
We have also identified a significant number of genes common between hESCs and MII oocytes. Such genes may provide the missing link between ESCs and MII oocytes and may serve as genetic resources to identify ESCs that have full potential for differentiation into an oocyte.
The results of the invention will facilitate a greater understanding as to the biological role of these genes and will expand enhance the understanding of the meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. The practical implications of compiling gene expression information on human gametes and embryos is enormous and will also bolster efforts to solve problems from infertility to degenerative diseases.
This invention in particular provides a transcriptome of 101 genes expressed by human oocytes. These genes and the orthologs of these genes are involved in the differentiation based on their specific expression in oocytes. Therefore, the introduction of one or more of these genes or the corresponding gene products should result in the transdifferentiation of a desired somatic cell into another cell type and/or lengthen the lifespan of said cell.
Particularly the introduction of at least one of said genes or incubation of a somatic cell or the nucleus or chromatin mass derived therefrom with a medium containing at least one of said gene products will result in the partial or complete dedifferentiation or reprogramming of a desired somatic cell into a pluripotent or multipotent cell., e.g., an adult stem cell or an embryonic cell type. Cells which transdifferentiate or dedifferentiate may be detected by screening by at least one marker that is specific for the particular cell type. For example, it is known that embryonic stem cells express certain genes such as Oct4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and alkaline phosphatase. Also, dedifferentiated cells or cells having enhanced lifespan may be detected based on an analysis of telomerase expression or telomere length. (Dedifferentiated cells will possess longer telomeres relative to parent differentiated cell). The subject methods may also be combined with the reprogramming procedures disclosed in US2005014258 published Jan. 20, 2005 and U.S. Pat. No. 7,253,334, issued on Aug. 7, 2007, both of which are incorporated by reference in their entirety herein. In the present invention the cells or the nucleus or chromatin mass derived therefrom with initially be contacted with a cocktail containing a significant number of the subject human oocyte specific gene products, e.g. 50, 40, 30, 20, 10 or more, and the cocktails which result in dedifferentiation will then be modified by removal of different gene products one at a time in order to determine the gene products which are essential for dedifferentiation or reprogramming, as well as the cocktail of constituents that achieves optimal results, i.e., production of cell with a stem cell phenotype. The optimal ratios and concentrations of these constituents and incubation time for the desired dedifferentiation will also be determined.
These transdifferentiated or dedifferentiated cells may be used e.g., for cell therapy or for study of the differentiation process. Additionally, these cells may be used to produce artificial tissues and organs by culturing said dedifferentiated cells in vitro e.g., in cell culture apparatus that are designed to facilitate the formation of desired cell structure and morphology. Additionally, these cells may be introduced into non-human animals as xenografted cells for example by injecting the dedifferentiated into desired organs. For example, dedifferentiated cells may be used to study the effect of dedifferentiated cardiac cells on damaged heart tissue to determine whether these cells promote the healing or regeneration process. Alternatively, dedifferentiated immune cells may be introduced into immunodeficient animals to assess whether this results in restoration of immune function.
The following references are cited in this application. The contents of these references is incorporated by reference herein.
This application claims priority to U.S. provisional Ser. No. 60/842,990 filed on Sep. 8, 2006. This application is incorporated by reference in its entirety herein.
| Number | Date | Country | |
|---|---|---|---|
| 60842990 | Sep 2006 | US |
