TWI493037B - Sbr and sbt non-embryonic stem cells and methods of making and using the cells - Google Patents

Description

SBR and SBT non-embryonic stem cells and methods for preparing and using the same

The present invention relates to novel stem cells of non-embryonic origin and uses thereof.

Totipotent stem cells, such as embryonic stem cells (ES cells), can differentiate into all cell lines in vivo and, when introduced into a test tube, can differentiate into most cell types. ES cells are derived from early mammalian embryos. Because of its versatility, it has great potential for treating degenerative or genetic diseases. However, ethical and supply considerations hinder the use of human ES cells for research and treatment. Non-embryonic pluripotent or pluripotent stem cells (such as tissues from adult or young animals) will evade this barrier. Although some stem cells have been obtained from such non-embryonic sources, some of these cells have limited developmental potential. Moreover, on the supply side, such cells are difficult to obtain and the amount of such cells is too limited to meet the needs of a meaningful clinical or research use. Furthermore, some of these cells develop into teratomas in vivo and are therefore not suitable for use in vivo. There is therefore a need for non-embryonic pluripotent or pluripotent stem cells that are safer, readily available, and have a rich source.

The present invention is based, at least in part, on the unexpected discovery that stem cell populations prepared from non-embryonic sources are pluripotent or pluripotent, can be obtained in very high yields, and do not develop into teratomas in vivo.

Therefore, one aspect of the invention features a method of making a pluripotent or pluripotent stem cell population. The method comprises: obtaining a plurality of blastomere-like stem cells (BLSCs), containing retinoic acid or transforming growth factor beta (TGF- β , transforming growth factor beta) The BLSCs are cultured in the medium, and the pluripotent or totipotent cells in the cultured cells are identified and enriched. These pluripotent or totipotent cells exhibit mRNA for GAPDH or β-actin. In other words, the amount of GAPDH or β-actin mRNA is detected by RT-PCR assay in the manner described in the Examples section below.

The pluripotent or pluripotent stem cells are typically from 1 to 15 microns in size, preferably from 1 to 10 microns in size, and more preferably from 1 to 5 microns in size. These dimensions are the size of the cells in the suspension or attached (i.e., suspended cells or attached cells).

The term "stem cell" refers to a cell that is capable of differentiating into many final, differentiated cell types. Stem cells can be pluripotent or pluripotent. Totipotent stem cells typically have the ability to develop into any cell type. Totipotent stem cells can be both embryonic and non-embryonic in origin. Pluripotent cells are typically cells that are capable of differentiating into several different types of terminally differentiated cells. Single-energy stem cells can only produce one cell type, but have the property of self-renewal to distinguish them from non-stem cells. Such stem cells are derived from a variety of tissues or organ systems including, but not limited to, blood, nerves, muscles, skin, intestines, bones, kidneys, liver, pancreas, thymus, and the like. According to the invention, stem cells are derived from adult or neonatal tissues or organs. BLSC (described in more detail below) is a group of non-embryonic stem cells in adult or young animals. These cells are pluripotent and have similar differentiation capabilities as embryonic stem cells. See WO2007/100845.

The cells obtained by culturing BLSCs in a medium containing RA were named SBR. In a specific embodiment, the medium contains 0.1 to 20 [mu]M RA and the BLSCs can be cultured for a total of 2 to 8 weeks. In another specific embodiment, the medium contains 1 to 15 [mu]M RA and the BLSCs are cultured for 2 to 8 weeks. In yet another embodiment, the medium contains 5 to 12 [mu]M RA and the BLSCs are cultured for 3 to 4 weeks. The pluripotent or pluripotent SBR cells thus prepared are 1 to 15 micrometers in size (such as 1 to 10, 1 to 5, 2 to 5, or 3 to 5 micrometers) and can form a sheet-like structure in suspension.

The cells obtained by culturing BLSCs in a medium containing TGF-β were named SBT. In a specific embodiment, the medium contains from 1 to 40 nM TGF-β and these BLSCs were cultured for 2 to 8 weeks. In another specific embodiment, the medium contains 2 to 20 nM TGF-[beta] and the BLSCs are cultured for 2 to 8 weeks. In yet another embodiment, the medium contains 5 to 12 nM TGF-[beta] and the BLSCs are cultured for 4 to 6 weeks. The pluripotent or totipotent SBT cells are 1 to 15 microns in size (such as 1 to 10, 1 to 5, 2 to 5, or 3 to 5 microns), have a circular shape and can form agglomerates.

Another aspect of the invention features a composition comprising a plurality of the above-mentioned cultured cells, wherein (1) is pluripotent or pluripotent, and (2) is 1 to 15 microns in size, (3) and exhibit mRNA of GAPDH or β-actin. The cells can be prepared by the methods described above. The composition may further contain RA or TGF-β. In a specific embodiment, the cells are CD10 ⁺ , CD90 ⁺ , CD105 ^+, and CXCR4 ⁺ . In another specific embodiment, the cells are negative for trypan blue staining. That is, these cells exhibit trypan blue exclusion. In another particular embodiment, the cultured cells containing such as CD66e ⁺ population of cells as well as the first CD66e ^- a second group of cells.

The cells are substantially pure. The term "substantially pure" when used to describe a stem cell or a cell derived from a stem cell (eg, a differentiated cell) means that the particular cell constitutes the majority of the cells in the formulation (ie, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%). In general, the substantially purified cell population constitutes at least about 70% of the cells in the formulation, typically about 80% of the cells in the formulation, especially at least about 90% of the cells in the formulation (eg, 95%, 97%, 99) % or 100%). Thus, the method of the present invention provides the advantage that a substantially pure population of specific types of cells (e.g., SBR or SBT cells) can be obtained without being contaminated by other cell types.

The above cultured cells can be used to express an exogenous recombinant polypeptide. Thus, such cultured cells are within the scope of the invention, each of which comprises a recombinant nucleic acid. The recombinant nucleic acid can encode a polypeptide and the cell can contain an mRNA encoding the polypeptide.

The cultured cells described above can be genetically manipulated such that they do not exhibit the β2-microglobulin gene or do not exhibit one or more of the major histocompatibility complex (MHC) genes. A protein of the protein that stimulates the anti-cell response of the T lymphocyte. Since these cells do not cause host rejection of the graft, they can be supplied as cells in an all-round manner.

In one aspect, the invention features a method of treating a degenerative disease in a patient. The method comprises administering an effective amount of the above composition to a patient in need of such treatment, the composition comprising one or more of the above pluripotent or totipotent cells. In a specific embodiment, at least one of the cells comprises a recombinant nucleic acid. The recombinant nucleic acid can encode a polypeptide and the cell can contain an mRNA encoding the polypeptide. Examples of degenerative diseases include diabetes, neurodegenerative diseases, arthritis, and cancer. Examples of neurodegenerative diseases include Parkinson's disease.

In another aspect, the invention features a method of treating an autoimmune disease in a patient. The method comprises administering an effective amount of the above composition to a patient in need of such treatment.

A patient to be treated for one of the above disorders can be identified by standard diagnostic techniques for a particular disorder. "Treatment" means administering a composition (eg, a cellular composition) to a patient at risk of developing the disorder or developing the disorder, for the purpose of the disorder, the symptoms of the disorder, and the disease secondary to the disorder. State or tendency to damage/disordered constitution to cure, alleviate, relieve, treat, delay its occurrence, prevent or relieve. By "effective amount" is meant the amount of a composition that produces a medically desirable result in a patient being treated. This treatment can be performed alone or in parallel with other drugs or therapies.

In yet another aspect, the invention features a candidate drug for the treatment of a degenerative disease. The method comprises the steps of contacting a test compound with the above composition or cells and determining the amount of expression of the polypeptide that is downregulated in the degenerative disease. If the amount of expression in the presence of the test compound is higher than the amount of expression in the absence of the test compound, it indicates that the compound is a candidate for the treatment of the disease. Examples of degenerative diseases include diabetes, neurodegenerative diseases, arthritis, cancer or autoimmune diseases. The amount of expression can be measured by either the amount of mRNA or the amount of protein.

In a further aspect, the invention features a method of introducing a heterologous nucleic acid into a patient. The method comprises the steps of: obtaining the above composition or cell, wherein At least one of the cells comprises a heterologous nucleic acid; and the cell is administered to a patient in need of the heterologous nucleic acid. The heterologous nucleic acid can encode a polypeptide. Once the cell is administered, the polypeptide will be expressed in the patient.

The term "heterologous" is a relative term that, when used to describe a portion of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found to have the same relationship in nature. For example, a nucleic acid produced by recombination typically has two or more sequences from unrelated genes that are synthetically arranged into a nucleic acid with a new function, such as a promoter from one source and a coding region from another source. The two nucleic acids are therefore heterologous to each other in their background. The recombinant nucleic acid, when added to a cell, is heterologous to the endogenous gene of the cell. Thus, in a chromosome, a heterologous nucleic acid introduces a non-native (non-naturally occurring) nucleic acid that is integrated into a chromosome, or a non-native (non-natively produced) extrachromosomal nucleic acid. In contrast, in the context of the present patent application, a natural position-changing fragment of a chromosome, when it comprises a native endogenous nucleic acid sequence, is not considered to be heterologous to the mutant cell. Similarly, a heterologous protein means that the protein comprises two or more subsequences (eg, "fusion proteins" in which they are not found to have the same relationship in nature, wherein the two subsequences are encoded by a single nucleic acid sequence). This protein can be produced by recombinant techniques.

The term "recombinant" when referring to a cell, nucleic acid, protein, or vector means that the cell, nucleic acid, protein, or vector has been introduced by introducing a heterologous nucleic acid or protein. A natural nucleic acid or protein is modified, or the cell line is derived from a cell through which the modifier is modified. Thus, for example, a gene expressed by a recombinant cell that is not found in the native (naturally produced) cell, or which exhibits a second set of a native gene, which can be expressed normally or abnormally, and is insufficiently expressed. Or not at all.

Included within the scope of the invention is a cell bank or library having a plurality of the foregoing compositions, each comprising a pluripotent or pluripotent cultured cell population. The cell can be a human cell or a non-human cell. The bank can obtain cells from a plurality of patients to obtain a plurality of embryonic leaf cell-like stem cell populations respectively; and identify the cell populations for each cell population One less predetermined feature; and the cell populations are sorted according to the at least one predetermined characteristic to prepare. When the library is prepared, the cell population can be further expanded. Examples of this feature include a patient's name, gender, physical condition (including genetic disease and MHC information).

A patient refers to a human or non-human animal. Examples of non-human animals include all vertebrates, such as mammals, such as non-human primates (especially high primates), dogs, rodents (such as mice or rats), guinea pigs, cats, Farm animals (such as horses, cattle, sheep, or pigs), and non-mammals, such as birds, amphibians, and reptiles. In a preferred embodiment, the patient is a human. In another embodiment, the patient is a test animal or an animal suitable for a disease mode.

The details of one or more specific embodiments of the invention are set forth in the following. The other features, objects, and advantages of the invention are apparent from the description and drawings.

It is generally believed that ES cells can be used to regenerate a variety of different cell types, such as neurons or glial cells in the brain, and thereby treat a variety of different degenerative diseases or tissue damage. However, ethical and supply considerations have hampered the use of ES cells. Stem cells other than embryonic sources, such as stem cells after childbirth (such as mesenchymal stem cells (MSCs)), are another promising alternative. Nonetheless, this alternative is not always accepted due to supply considerations and limitations on proliferation/differentiation.

The present invention relates to stem cell populations, SBR cells, or SBT cells prepared from non-embryonic sources. Just like ES cells, these cells are also pluripotent or pluripotent. More importantly, these cells can be obtained in very high yields and do not develop into teratomas in vivo . These cells can thus be used to regenerate differentiated cells to treat a variety of different degenerative diseases or tissue damage. As shown in the examples below, SBR and SBT cells can be easily manufactured, maintained and propagated in vitro , and conventional techniques are used to induce differentiation. In addition, after transplantation of the cells into an animal patient (eg, a mouse), there are no mitotically activated cells, teratomas, or malignant growth. Based on these advantages, these cells present an alternative to other stem cells.

In a preferred embodiment of the invention, the SBR and SBT cell lines are prepared from a germ cell-like stem cell population (BLSCs). BLSCs are a group of non-embryonic stem cells in adult or young animals. These cells are pluripotent and have similar differentiation capabilities to embryonic stem cells. See WO2007/100845. BLSCs contain a normal set of chromosomes, and BLSCs are lineage-uncommitted and can form all body (non-reproductive) cells of the body. These cells can also form reproductive gamete sperm and/or eggs, as well as cells, as well as embryonic tissues and fetal parts of the placenta. The cells are susceptible to response to lineage inducing agents, proliferative agents, and differentiation inhibitors. On the other hand, the cells are less reactive to progression agents. Similar to ectodermal-like stem cells, BLSCs have no contact inhibition when cultured to fullness, but when they are supplied in a desirable nutrient supply, they form multi-layered cells. Floor. BLSCs do not exhibit phenotypic expression markers representing precursor or differentiated cells, germline lineage stem cells, or ectodermal-like stem cells. Conversely, BLSCs exhibit general and specific embryonic lineage markers, such as the embryonic stem cell markers CD66e, HCEA, CEA, and CEA-CAM-1. BLSCs are often static in mature organizations. However, when the above tissues are damaged, the BLSCs are activated and differentiated to repair the damaged tissue.

Compared to other stem cells, BLSCs can achieve relatively high yields from a post-partum tissue. For example, BLSCs can be obtained from blood in a yield exceeding 2 x 10 ⁸ per ml of blood. On the other hand, since BLSCs are static and do not express genes, the use of such cells for research and therapeutic purposes is also limited.

Unexpectedly, culturing BLSCs with certain chemicals produces pluripotent or pluripotent stem cells that can express endogenous genes and heterologous genes. The two pluripotent or pluripotent stem cell populations are the SBR cell population and the SBT cell population. will BLSCs were cultured in medium containing RA and TGF-β to obtain SBR cell population and SBT cell population.

BLSCs can be prepared by the methods described in the Examples section below, or by the methods described in WO2007/100845. In general, the cells can be isolated from a variety of tissues of adult or young animals, including blood, bone marrow, and skeletal muscle. To confirm that the isolated cells are indeed BLSCs, some of the features available for testing include (1) cell size in suspensions of less than 1 micron; (2) cell surface markers such as CD66e ⁺ ; and (3) Blue staining was positive. Cell surface marker antibodies such as CD66e can be used. It can be applied by labeling, such as: fluorescein isothiocyanate, phycoerythrin, or quantum dots. BLSC, which is CD66e ⁺ , can be further enriched by flow cytometry (Fig. 1 to Fig. 2).

This enriched cell population is then tested using standard techniques. To confirm the differentiation potential of such cells, such cells can be induced to form by conventional techniques, such as: neuronal-glial cells, bone cells, and adipocytes. For example, the cells can be subcultured and cultured to confluence, transferred to an osteogenic medium or a lipogenic medium, and cultured for a suitable period of time (eg, three weeks). The ability to differentiate into bone formation can be assessed by mineralization of calcium accumulation, which can be observed by von Kossa staining. The intracellular oil droplets were stained by Oil Red O and observed under a microscope to detect the formation of adipogenic differentiation. For neuronal differentiation, the cells can be cultured in a neurogenic medium for a suitable period of time (eg, seven days), then treated with serum depletion and cultured with β-mercaptoethanol. . Upon differentiation, the cells present a refractile cell body with an extended axon-like structure to form a network. Immunocytochemical staining with lineage-specific markers can further guide the differentiation of nerves. Examples of such markers include neuron-specific third class β-tubulin (Tuj-1), neurofilament, and GFAP.

In addition, the identity of the isolated cells can be confirmed by the fact that BLSC does not have a contact inhibition effect. To this end, the isolated cells can be cultured to fullness. Under these conditions, BLSCs can form spherical cell aggregates, multiple confluent layers, or mesh-net structures. In contrast, CD42 ⁺ cells are unable to form the aforementioned structures, such as cell aggregates.

The BLSCs confirmed by the above can be further cultured in a non-differentiating medium with a population doubling of more than 10, 20, 50, or 100 generations, and no spontaneous differentiation, aging, and Morphological changes, increased growth rates, or changes to signs of ability to differentiate into neurons. These cells can be stored in a standard manner prior to use.

To prepare SBR cells, the BLSCs can be cultured for 2 to 8 weeks in an environment containing 0.1 to 20 μM RA. In a preferred embodiment, the BLSCs are cultured for 2 to 8 weeks in an environment containing 1 to 15 μM RA, or more preferably, for 3 to 4 weeks in an environment containing 5 to 12 μM RA. The pluripotent or pluripotent cell population thus prepared has a size of 1 to 15 microns and can form a sheet-like structure in the suspension. Any commercially available RA suitable for cell culture can be used.

To prepare SBT cells, the BLSCs can be cultured for 2 to 8 weeks in an environment containing 1 to 40 nM TGF-β. In a preferred embodiment, the BLSCs are cultured for 2 to 8 weeks in an environment containing 2 to 20 nM TGF-β, or more preferably, cultured in an environment containing 5 to 12 nM TGF-β. 6 weeks. The pluripotent or pluripotent cell population is 1 to 15 microns in size, has a rounded shape, and can form agglomerates. Although any type of TGF-β can be used, high purity TGF-β is still preferred. Examples of TGF-β include mammalian TGF-β (e.g., human TGF-β), or TGF-β having substantially the same biological activity as mammalian TGF-β. Both naturally occurring TGF-β and genetically engineered TGF-β can be used. The TGF-β obtained by the DNA recombination technique may have the same amino acid sequence as the naturally produced TGF-β, or a functional equivalent thereof. A "functionally equivalent" refers to a polypeptide derivative of a naturally occurring TGF-β, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or a combination thereof. The term "TGF-β" also encompasses chemically modified TGF-β. Examples of the chemically modified TGF-β include structurally altered TGF-β, addition or deletion of its sugar chain, and TGF-β bound by a compound such as polyethylene glycol.

The SBR cells and SBT cells can be distinguished from and confirmed by BLSCs based on their size, contact inhibition, trypan blue stain rejection (eg, trypan blue staining negative). BLSCs are smaller (less than 1 micron) than SBR cells and SBT cells, and lack contact inhibition or trypan blue stain rejection. The SBR cells and SBT cells can also be confirmed by flow cytometry according to relevant cell markers such as CD10 ⁺ , CD90 ⁺ , and CXCR4 ⁺ , or other standard analytical methods such as RT-PCR.

The SBR cells and SBT cells thus prepared can be further tested in the same manner as the aforementioned BLSCs, or other standard techniques of the prior art, to test their pluripotency and pluripotency. Under appropriate conditions of induction, the cells can be differentiated in vitro, such as: adipocytes, chondrocytes, and cell populations of the osteogenesis lineage. Furthermore, under the aforementioned induction conditions, the cells have the ability to dedifferentiate into glial cells.

Versatility and versatility can be tested in vivo. For example, when a rat with ischemic brain receives intracerebral transplantation of SBR cells or SBT cells in the brain, its neurological function is significantly improved compared to a control group treated with a placebo. This result indicates that SBR cells or SBT cells administered in the brain can enter the brain, survive, migrate, and improve the functional recovery rate after stroke. In fact, these transplanted cells can differentiate into glial cells (GFAP+), neurons (Nestin+, MAP-2+, and Neu-N+), and vascular endothelial cells (vWF+) in the ischemic brain to enhance The effect of neuroplasticity. The neuronal activity of the cerebral cortex can be assessed by the hydrogen proton magnetic spectroscopy ( ¹ H-MRS, Proton MR spectroscopy), and the neuronal activity in the cerebral cortex of the transplanted group is also increased significantly compared to the control group. In addition, the regulation of neurotrophic factors was also found to be significantly increased in the ischemic cerebral hemisphere of the transplantation group.

The SBR cells and SBT cells confirmed by this can be further cultured in a non-differentiating medium at a cell doubling rate of 10, 20, 50, or 100 generations, and no spontaneous differentiation or aging therein. , morphological changes, increased growth rate, or altered to have the ability to differentiate into neurons. These cells can be stored in a standard manner prior to use.

The terms "proliferation" and "amplification" are used interchangeably herein to refer to a cell, which refers to an increase in the number of cells of the same species by division. The term "differentiation" refers to the transformation of a particular function during cell development, for example, the cell acquires one or more morphological features and/or functions that differ from the original cell type. The term "differentiation" encompasses lineage commitment and terminal differentiation processes. Differentiation can be assessed, for example, by using immunohistochemistry or other steps known to those skilled in the art to monitor the presence or absence of lineage markers. Differentiating cells from progenitor cells may be, but not necessarily, the same germ layer or tissue as the stem cell source. For example, neural precursor cells and muscle precursor cells can differentiate into a hematopoietic cell lineage.

The terms "lineage decision" and "specialization" are used interchangeably herein to refer to the process by which a stem cell develops into a precursor cell that determines the type of cell differentiation that forms a particular restricted range. Priorized cells have been directed to have the ability to self-renew or divide cells.

The term "terminal differentiation" refers to the final differentiation of a cell into a mature, fully differentiated cell. For example, neural precursor cells and muscle precursor cells can differentiate into a hematopoietic lineage that is terminally differentiated into a mature blood cell belonging to a particular cell type. Generally, terminal differentiation is associated with disrupting the cell cycle and stopping proliferation. The term "progenitor cell" as used herein refers to a cell that has been determined to be a particular cell lineage and which is developed into a population of cells of the lineage by a cascade of cell divisions. An example of a precursor cell can be a myoblast that can only differentiate into a single type of cell, but is not fully mature or fully differentiated by itself.

1. General use

The SBR cells and SBT cells of the present invention can be applied to many levels. It can be used to treat degenerative or hereditary diseases and to eliminate the ethical considerations of manipulating human embryos.

For this purpose, BLSCs can be isolated in vivo from patients (eg, important functional genes lacking the proper development of tissues or organs). After the SBR cells and SBT cells are produced from BLSC, a nucleic acid vector capable of encoding a functional gene can be transferred into the cells. The vector can be transferred into cells by a number of means, including calcium phosphate coprecipitation, DEAE dextran vector transfection, liposome transfection, electroporation, microinjection, or viral media. A method which does not affect the pluripotency of the cell is preferred. A detailed description of such techniques can be found in U.S. Patent Nos. 7,422,736 and 5,591,625, and U.S. Patent Application No. 20020127715. After the functional genes are delivered into the cells, the cells are then transplanted into the patient by conventional techniques. Since the cell line is produced from the patient, this treatment does not cause immune rejection.

In addition, universal donor cells can be made using SBR cells and SBT cells prepared from a healthy individual. Methods for preparing universal donor cells are well known in the art, and such methods for preparing universal SBR cells and SBT cells will be described in detail below.

Under appropriate conditions, the transplanted SBR cells and SBT cells can develop into a functional tissue or organ. In order to promote its development, the patient may be administered a factor that induces cell development. These factors can be small molecule compounds, peptides, and nucleic acids. Examples include, but are not limited to, transforming growth factor beta, bone morphogenetic protein, and nerve growth factor.

These SBR cells and SBT cells are also useful for the study of the developmental or differentiation mechanisms of lineage development and differentiation. These cells are used as a model system to identify conditions that induce pluripotent pluripotent stem cells to become a particular tissue or organ. In addition, conventional differential cDNA gene screening methods can be used to isolate genes involved in development. See, for example, Shen M. et al., Development, 124: 429-42, 1997. Such cells can be utilized which have been induced to develop into a particular lineage, such as the aforementioned glial cell line, to prepare a cDNA library. This library can be used to isolate and study genes with differential performance. These isolated genes can be further used to study their roles in their respective stages. Related techniques are well known techniques. See, for example, U.S. Patent No. 7,422,736 and U.S. Patent Application No. 20060035373. Such SBR cells and SBT cells can also be used for hair growth as an organ or a replicating animal using conventional methods. See, for example, Campbell K. et al., Nature, 380: 64-66, 1996. Therefore, these cells are of great value to the pet and livestock industry and can be used to preserve endangered animals.

2. Screening method

The aforementioned stem cells can be used in a screening test to identify a drug that affects a particular cell type in a particular manner, i.e., the drug can be used to treat a disease associated with that cell type.

Accordingly, one aspect of the invention relates to a method of identifying a formulation by contacting a test reagent with a cell to find an agent that alters the function of undifferentiated SBR cells and SBT cells. The test preparation is an agent that alters the function or gene expression of the cells, if the function or gene expression of the cells is altered in the presence of the test reagent. The term "test formulation" refers to any molecule that is used to detect its ability to alter cellular function or gene expression. Although the method is generally used as a screening method to identify a molecule that was previously unknown but has a desired activity, the screening method of the present invention can also be used to identify a formulation known to possess a particular activity.

The function may be the expression of a gene, which generally refers to the performance (or no performance) in the cell, and the amount of expression of the agent by increasing or decreasing a gene of expression (eg, reducing the amount of expression of CD66e), or by The performance of an unexpressed gene (eg, the expression of an antigen that induces a particular lineage) in a cell is turned on to alter this function.

In one embodiment, the formulation that affects cellular function is a formulation that induces cell differentiation and produces differentiated cells. Such differentiated cells may be pluripotent human stem cells (eg, hematopoietic stem cells), or may be final differentiated cells (eg, muscle cells, nerve cells, blood cells, connective tissue, or epithelial cells). This method, by itself, can be used to identify a preparation that can induce differentiation of SBR cells and SBT cells into cells that ultimately differentiate, such as islet beta cells, hepatocytes, cardiomyocytes, skeletal muscle cells, or any other cell type. The formulation or compound identified by this method can be used to treat degenerative diseases, cancer or immune diseases.

The amount of expression can be determined by the amount of mRNA expression or protein expression. The method of measuring the amount of mRNA expression in the same manner is a well-known technique. To measure mRNA expression, cells can be lysed, and the amount of mRNA present in the cell lysate, whether purified or not, can be achieved by, for example, a hybridization assay (using detectable DNA or RNA for specific genes) Needle), as well as quantitative or semi-quantitative RT-PCR (using appropriate specific gene primer pairs). Alternatively, quantified or semi-quantized in situ hybridization assays can be used for tissue sections, or undissolved cell suspensions, which can be labeled with a detectable (eg, fluorescent or enzymatic) DNA or RNA probe. Other mRNA quantification methods include the RNA protection assay (RPA) method, as well as gene expression sequence analysis methods (SAGE), and array-based techniques.

The method of measuring the amount of protein expression in the present is a well-known technique. Some of these antibodies (eg, monoclonal antibodies or polyclonal antibodies) can be specifically bound to a target protein. In such assays, the antibody itself or a second antibody that binds thereto is labeled as a detectable. Additionally, the antibody can bind to biotin. Its presence can be measured by detectable calibrated avidin (a peptide system bound to biotin). A combination of these methods (including the "Multilayer Sandwich Structure" test) can be used to enhance the sensitivity of the method. Some protein measurement tests (eg ELISA or Western blot) can be applied to body fluids or cell lysates, or other (eg immunohistochemical methods or fluorescent flow cytometry) for tissue sectioning or undissolved Method of cell suspension. Suitable calibrations include radionuclides (eg ¹²⁵ iodine, ¹³¹ iodine, ³⁵ sulphur, ³ sputum or ³² sulphur), enzymes (eg alkaline phosphatase, horseradish catalase, luminescent enzyme, or beta-galactoside) enzymes), fluorescent / luminescent agent (such as: fluorescein, pink fine salt, phycoerythrin, GFP, BFP, and by the Quantum Dot Corporation, Palo Alto, CA Qdot ^TM supplied by the nanoparticles). Other applicable methods include quantitative immunoprecipitation analysis, or complement fixation assays.

A test preparation may be any type of molecule, for example, a polynucleotide, a peptide, a phenopeptide, a peptidomimetic may be a vinylogous peptoids, a small organic molecule or the like, And in various ways to change the function of SBR cells and SBT cells. For example, the test formulation can be altered by extracellular binding to a receptor present on the cell surface, thereby altering a function that is mediated by a ligand that normally binds to and acts on the receptor. In addition, the test formulation can change a function passively or by an active transport mechanism to cross the cell membrane and act within the cell.

The peptide test formulation can be a polymer of any amino acid or amino acid analog and can vary from three to four residues to hundreds or thousands of residues. The peptide test preparation can be prepared by chemical synthesis, or by protein purification, followed by proteolysis, and further purified by chromatography or electrophoresis if required, or can be expressed by encoding a polynucleotide. . The peptide test formulation can be a known peptide, such as a naturally occurring peptide, but can also differ from the naturally occurring sequence, for example, comprising one or more amino acid analogs.

A polynucleotide preparation may be a sequence of two or more deoxyribonucleic acids or ribonucleic acids linked to each other by a phosphodiester bond. It may be RNA or DNA, which may be a gene or a protein thereof, a cDNA, an RNAi reagent, a synthetic polyoxyribonucleic acid sequence, or the like, and may also be single-stranded or double-stranded, or may be It is a DNA/RNA hybrid. It can be a naturally occurring nucleic acid molecule isolated from a cell, or a synthetic molecule obtained by, for example, chemical synthesis or an enzyme method (such as polymerase chain reaction). . In various embodiments, the polynucleotides of the invention may comprise a nucleoside or nucleotide analog, or a backbone linkage of a non-phosphodiester linkage. Such nucleotide analogs are well known in the art and are commercially available, and polynucleotides comprising such nucleotide analogs are also the same (Lin et al., Nucl. Acids Res. 22: 5220-5234, 1994; Jellinek et al., Biochemistry 34: 11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15: 68-73, 1997).

A polynucleotide test preparation can be contacted with SBR cells and SBT cells, or transferred to SBR cells and SBT cells by methods or techniques described herein. In general, and not necessarily, when the polynucleotide is transferred into a cell, it can directly affect its function, or affect its transcription or translation, or both. For example, the polynucleotide can encode a peptide test preparation that can be expressed intracellularly and affect the function of the cell. A polynucleotide may also be, or encode, a counter-strand molecule, a ribozyme, or a three-strand synthetic reagent that can be designed to target one or more specific target nucleic acid molecules.

Candidate formulations or compounds to be screened (e.g., proteins, peptides, peptidomimetics, peptidomimetics, antibodies, small molecules, or other drugs) can be obtained by any of a variety of routes in the combinatorial library methods known in the art. These libraries contain: a peptide library, a peptidomimetic library (a library of peptides with novel peptides, which are resistant to enzymatic degradation, a novel, non-peptide structure); space-addressable parallel solid phase or solution phase library (spatially) The addressable parallel solid phase or solution phase libraries); the synthetic molecular library obtained by the unwinding or affinity chromatography; and the "single bead single compound" molecular library. See, for example, Zuckermann et al . 1994, J. Med. Chem. 37: 2678-2685; and Lam, 1997, Anticancer Drug Des. 12: 145. Examples of synthetic methods for molecular libraries can be found, for example, in DeWitt et al ., 1993, PNAS USA 90:6909; Erb et al ., 1994, PNAS USA 91:11422; Zuckermann et al ., 1994, J. Med. Chem. 37:2678; Cho et al ., 1993, Science 261:1303; Carrell et al ., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al ., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al ., 1994 J. Med. Chem. 37:1233. Compounds of the molecular library can be presented in liquids (eg, Houghten, 1992, Biotechniques 13: 412-421), or on beads (Lam, 1991, Nature 354: 82-84), wafers (Fodor, 1993, Nature 364: 555-556), Bacteria (US Patent No. 5,223,409), Spores (US Patent No. 5,223,409), plastids (Cull et al ., 1992, PNAS USA 89:1865-1869), or phage (Scott and Smith 1990, Science 249) : 386-390; Devlin, 1990, Science 249: 404-406; Cwirla et al ., 1990, PNAS USA 87: 6378-6382; Felici 1991, J. Mol. Biol. 222: 301-310; 5,223,409).

3. Treatment of degenerative diseases

Within the scope of the present invention is a method of treating a degenerative disease, soothing the symptoms of the disease, or delaying the onset of the disease in a patient. The patient's condition or disease can be diagnosed by standard techniques to identify the person in need of treatment. The method of treatment requires administration of an effective amount of the aforementioned SBR cells or SBT cells to a patient in need of treatment.

A degenerative disease is a disease in which the function or structure of an infected tissue or organ gradually deteriorates over time, regardless of genetic defects, injuries, lack of proper cell differentiation (eg, diseases of cell proliferation), and normal body Wear, or lifestyle choices. Examples of degenerative diseases include neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis). ), other neurological diseases (including transverse myelitis, nerve de-sheathing after trauma to the brain or spinal cord, acute brain damage, head trauma, spinal cord damage, peripheral nerve damage, ischemic brain) Impaired, hereditary myelin disorders in the central nervous system, epilepsy, asphyxia in the perinatal period, asphyxia, anoxia, epileptic reabsorption, Shy-Drager's disease, autism and stroke), cancer or related cancer therapy Situation (eg chemotherapy); metabolic diseases (eg diabetes, Niemann's disease); autoimmune or inflammatory related diseases (eg erythema, iinflammatory bowel disease, prostatitis, bone) Arthritis, osteoporosis, rheumatoid arthritis, lupus, diabetes, and asthma), eye diseases (eg glaucoma, retinitis pigmentosa, Norrie's disease, and macular degeneration); heart or circulatory system Diseases (eg, atherosclerosis, heart failure, myocardial infarction, and cardiovascular disease); blood diseases such as: Wiscott Aldrich's syndrome; muscular dystrophy; gastrointestinal disease; kidney disease; liver disease; lung disease, kidney disease (such as Edison's disease), burns caused by burns or strokes, including: damaged tissue (such as: muscle damage, aging damaged cells and aging damaged tissue), and age-related conditions (such as: hair loss) , male baldness and round baldness, viral conditions (eg, hepatitis C virus infection, and acquired immunodeficiency syndrome), and others may be repaired by organ transplantation or stem cells, reborn or improved, and/or associated with the disease Related symptoms. The method of the invention can be used to treat erectile dysfunction, and orthopedics, or breast implants in women.

4. Treatment of Parkinson's disease and other neurodegenerative diseases

Within the scope of the present invention is a method of treating damage to a brain or central nervous system tissue or alleviating the symptoms of a disease in a patient. This method includes identifying patients who are suffering from or presenting a potential risk of developing damage to brain tissue. This patient can be a human, or non-human mammal, such as a cat, dog, or horse. Examples of brain tissue damage include ischemia by the brain (eg, chronic stroke), or neurodegenerative diseases (eg, Parkinson's disease, Alzheimer's disease, spinocerebellar disease, or Huntington's disease) The damage caused. The patient's condition or disease can be diagnosed by standard techniques to identify the person in need of treatment. The method of treatment requires administration of an effective amount of the aforementioned SBR cells or SBT cells, or an active ingredient/compound, to a patient in need of treatment.

The efficacy of this cell can be measured by standard methods. For example, to confirm its role in promoting cerebral angiogenesis, a patient can be examined by standard brain imaging techniques before and after treatment, such as computed tomography (CT), Doppler ultrasound imaging (DUI, Doppler ultrasound imaging, magnetic resonance imaging (MRI), and hydrogen proton magnetic resonance spectrum ( ¹ H-MRS). For example, the hydrogen proton magnetic resonance spectrum ( ¹ H-MRS) represents a non-invasive method for obtaining biochemical information related to brain metabolic activity (Lu et al., 1997, Magn. Reson. Med. 37, 18- twenty three). This technique can be used to assess metabolic changes associated with cerebral ischemia before and after stem cell transplantation. For example, it can be used to study the concentration of N-acetylasparate in the brain, which is an indicator of brain neuron integrity. Although the NAA redistribution and sequestration in neuronal fragments limits its use as a certain amount of neuronal markers, the decrease in brain NAA concentration during cerebral ischemia can still be used as an indicator of neuronal loss or dysfunction. (Demougeot et al., 2004, J. Neurochem. 90, 776-83). Therefore, the NAA concentration measured by the hydrogen proton magnetic resonance spectrum ( ¹ H-MRS) can be used as an effective indicator to track the effect of post-ischemic stem cell transplantation.

5. Cancer

The aforementioned SBR cells or SBT cells can also be used to treat diseases of cancer and other cell proliferation. A cell proliferative disorder is characterized by an uncontrolled, spontaneous cell growth (including malignant and non-malignant growth). The term "cancer" refers to a disease of this type characterized by uncontrolled cell growth, invasion, and sometimes metastasis. The ability of a cancer cell to grow spontaneously, for example, the state or condition of an abnormally rapid proliferating cell. The term is intended to encompass all types of cancerous growth, or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues, or organs, regardless of their histopathological type, or stage of invasion. Examples of cancer include, but are not limited to, cancer and sarcoma, such as: leukemia, sarcoma, osteosarcoma, lymphoma, melanoma, glioma, pheochromocytoma, liver cancer, ovarian tumor, skin cancer, testicular cancer, stomach cancer , pancreatic cancer, kidney cancer, breast cancer, prostate cancer, colorectal cancer, head or neck cancer, brain cancer, esophageal cancer, bladder cancer, renal cortical cancer, lung cancer, bronchial cancer, endometrial cancer, nasopharyngeal cancer, Cervical cancer or liver cancer, and other cancers of unknown origin.

6. Gene therapy

The foregoing cells and methods can be used in a variety of different conventional gene therapy methods. Gene therapy includes ex vivo and in vivo techniques. In particular, the aforementioned stem cells can be genetically engineered in vitro by an oligonucleotide modulator or a nucleic acid molecule encoding the regulator, and the engineered cells are provided to a patient to be treated. The cell culture can be formulated to administer a patient, for example, by isolating the cell (eg, by mechanical separation) and attaching the cell to a pharmaceutically acceptable carrier (eg, phosphate buffer) well mixed. Alternatively, the cells can be cultured in a suitable biocompatible carrier and implanted into a patient. Such engineered cells are generally autologously derived to avoid allogeneic or xenogeneic rejection. These ex vivo methods are all known techniques.

Such cells can be engineered by administering oligonucleotides or nucleic acid molecules by conventional techniques. For example, oligonucleotides and other nucleic acid molecules can be administered by direct injection of a "naked" nucleic acid molecule (Felgner and Rhodes, (1991) Nature 349:351-352; US Pat. No. 5,679,647). Or a nucleic acid molecule can be formulated in a composition comprising one or more components that promote absorption of the nucleic acid molecule by the cell, which component can be a saponin (see, e.g., U.S. Patent No. 5,739,118), or a cationic polyamine. (See, for example, U.S. Patent No. 5,837,533); or by microprojectile bombardment (for example, by using a "gene gun"; Dupont Pharmaceuticals Biolistic); or by using the nucleic acid molecule as a lipid, cell Coating with a surface acceptor, or a transfection agent; or coating the nucleic acid molecule in a liposome, microparticle, or microcapsule; or by administering a nucleic acid molecule linked to a peptide, the peptide Known to enter the nucleus; or by administering a nucleic acid molecule linked to a ligand that is susceptible to the pinocytosis of the receptor vector, which can be used to calibrate the receptor for specific expression Cell type.

A nucleic acid-ligand complex can be formed, wherein the ligand comprises a fusion virus peptide to disrupt the endosome, which can protect the nucleic acid from being degraded by lysosomes; or the nucleic acid molecule can be used as a target for cell specific absorption And targeting a specific receptor for in vivo performance. In addition, an efficient method of introducing, expressing, and accumulating anti-strand oligonucleotides is described in U.S. Patent No. 6,265,167, which causes the anti-oligonucleotide to hybridize to a nucleus mRNA in the nucleus. It is also possible to prevent the anti-strand oligonucleotide from being processed or transported to the cytoplasm. The present invention also contemplates the incorporation of the nucleic acid molecule into a cell, and subsequent chimerization of the host cell DNA by conventional homologous recombination techniques.

The polynucleotide may also be embedded in a suitable expression vector. Some well-known vectors for gene therapy applications are known (see, for example, Viral Vectors: Basic Science and Gene Therapy, Eaton Publishing Co. (2000)).

The performance vector can be a plastid vector. The method of generating and purifying plastid DNA is fast and simple. In addition, when a discrete entity elimination genotoxicity issue occurs that may lead to chromosomal integration, plastid DNA generally does not integrate into the genome of the host cell, but is maintained at the episomal location. A variety of commercially available vectors are now available, as well as those comprising Escherichia coli and Bacillus subtilis, many of which are specifically designed for use in mammalian systems. Examples of plastids that may be used in the present invention include, but are not limited to, eukaryotic expression vectors pRc/CMV (Invitrogen), pCR2.1 (Invitrogen), pAd/CMV, and pAd/TR5/GFPq (Massie et al. (1998) Cytotechnology 28: 53-64). In an exemplary embodiment, the system is pRc/CMV, pRc/CMV2 (Invitrogen), pAdCMV5 (IRB-NRC), pcDNA3 (Invitrogen), pAdMLP5 (IRB-NRC), or PVAX (Invitrogen).

The expression vector can be a viral vector. Examples of viral vectors include, but are not limited to, viral vectors derived from retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses that are unable to self-replicate. Retroviral vectors, as well as adeno-associated viral vectors, are among the first choices in current recombinant gene delivery systems, which are useful for the in vivo delivery of exogenous oligonucleotides, or genes, particularly to humans. These vectors allow the gene to efficiently enter the cell and allow the delivered nucleic acid to be stably integrated with the host's chromosomal DNA. One of the main prerequisites for the use of retroviruses is the safety of the use of such viruses, with particular attention to the possibility of wild-type viruses spreading in the cell population. The retroviral vector may be derived from a retrovirus, including, but not limited to, Moloney murine leukemia virus, pancreatic necrosis virus, retrovirus (such as Rous sarcoma virus, Harvey's murine sarcoma virus, Avian leukosis virus, gibbon leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and breast tumor virus. Specific retroviruses include pLJ, pZIP, pWE, and pEM, which are known to the public. All know.

7. Cell bank

One feature of the invention resides in a stem cell bank or library that facilitates and systematic access to different stem cell lines. The stem cells in the bank or library are derived from the aforementioned BLSC, SBR cells, or SBT cells, which may be derived from a healthy individual, or a patient having a known disease state or the symptom is for a user (eg, a researcher). Said to be an important treasure. Cell banks or libraries having cells differentiated from the aforementioned stem cells are also within the scope of the invention. Examples of the cells differentiated from the stem cells include brain cells, neuronal cells, astrocytes, glial cells, T cells, B cells, chondrocytes, bone cells, islet cells, fat cells, heart cells, liver cells, Kidney cells, lung cells, muscle cells, and eye cells. The patient can be a human or non-human vertebrate. The stem cells can be derived from any mammalian species, such as humans, mice, rabbits, cows, pigs, and the like.

Cell lines in banks or banks are classified according to predetermined characteristics, including phenotypic information, morphological features, differentiated morphology, blood type, major histocompatibility complex, donor disease status, or genotype information (eg, : One of a single nucleic acid sequence of a single nucleotide nucleated polymorphism (SNP) is associated with a gene, a gene body or a mitochondrial DNA). The cell lines are maintained in a suitable environment (generally by freezing) to maintain the stem cells alive and still function. Classification may include establishing a centralized record of characteristics of each cell population, such as, but not limited to, an assembled written record, or a computer database into which information is entered. Essentially, this particular embodiment relates to the preparation of a stem cell bank. The stem cell bank helps the user to screen out a number of samples for specific stem cell samples that are suitable for the user's needs. Moreover, another embodiment of the present invention is directed to a stem cell bank comprising a plurality of stem cell samples from separate sources, and the samples are characterized and classified based on at least one predetermined feature. Another embodiment relates to a method of establishing a stem cell bank comprising stem samples collected from a plurality of sources; classifying the samples according to at least one predetermined characteristic, and storing the cells under specific conditions such that It survives.

In the context of the present invention, a stem cell banking system comprising a plurality of stem cell populations is placed in individual containers under specific conditions to allow the stem cell population to survive; a computer database comprising at least one processing module, a display and a And a storage medium comprising at least one characteristic information for each stem cell group; and at least one code module, wherein when the user issues an instruction, the generated information can be browsed on the display by the code module. In a particular embodiment, one of the features of the invention resides in that the stem cell population of the stem cell banking system is derived from stem cells of a patient having a disease condition. The condition can include the aforementioned degenerative diseases. Stem cells collected from patients with different diseases are characterized. These features are entered into a computer database. Moreover, or alternatively, the cell lines are characterized based on a particular phenotype but are not necessarily associated with a disease condition. For example, hepatocytes can be characterized based on their ability to metabolize certain classes of compounds, such as caffeine, alcohol, pharmaceutical preparations, etc., to study the genetic nature of these different metabolic abilities, or the underlying physiology associated with them. Other cell types can be characterized based on their functional and/or morphological phenotype.

In some embodiments, cells differentiated from SBR cells or SBT cells can be susceptible to conditions affecting differentiation or rejection by introducing an engineered vector, or other genetic material. Rejection differentiation involves manipulating a cell to have a characteristic of a less differentiated cell.

The stem cell bank of the present invention can be screened out as a formulation or compound useful for treating degenerative diseases, cancer or immune diseases by the foregoing means. This library is suitable for use in high speed screening systems and can help identify agents that are particularly effective for a particular patient. For a high speed screening system, the stem cells can be introduced into a well of a porous disk or onto a slide or microchip and contacted with the test formulation. Generally, the cells are organized in an array, especially an addressable array, which facilitates manipulation of the cells and solutions by the robot, and monitoring of such cells, such operations Especially related to the function being tested. The advantage of using a high yield form is that many test reagents can be tested simultaneously, and, if desired, the reaction of the control group can be reacted under exactly the same conditions as the test conditions. Thus, the screening method of the present invention provides a means for screening from one, some or a large number of test formulations to identify a formulation that alters stem cell function, for example, the formulation comprises: differentiating the cell into a desired cell type Or can avoid spontaneous differentiation, such as: regulatory molecules that can maintain high levels of performance.

8. Universal donor cells

The aforementioned stem cells can be engineered to produce tissue antigen compatible donor cells or tissues for transplantation. The goal of transplantation and cell therapy is to successfully replace a tissue or organ that is functional with a donor tissue or organ that is depleted. However, in order to achieve a successful transplant, two barriers need to be overcome: suitable donor tissue or organ acquisition, and immune rejection. Treatment of depleted tissues or organs and immune rejection is limited by the limited number of acceptable donors and the simultaneous administration of toxic immunosuppressive drugs in long-term immunosuppressive therapies. Current and experimental transplant methods rely primarily on relative donors and other very small numbers of allogeneic or xenogeneic donors. The aforementioned genetically engineered stem cells can be used to overcome these limitations.

More specifically, the stem cells described herein can be engineered using genetic engineering operations such that they do not exhibit a second class of major histocompatibility complex molecules (class II MHC molecules) on the cell surface. Preferably, the cells are engineered such that they do not exhibit primary and second major histocompatibility complex molecules on substantially all cell surfaces. As used herein, the term "does not express" means that the reaction is not caused by insufficient performance on the surface of the cell, or that the expressed protein is defective and does not cause a reaction.

The major histocompatibility complex molecule refers to HLA molecules, specifically to the first classes of HLAA, B and C, and the second class of HLA DP, DQ, and DR, and subclasses. This term is often interpreted to refer to a human major histocompatibility complex, but the equivalent major histocompatibility complex genes from other donor species can be derived from this article, for example, if the cell source is a pig, the term is used. HLA refers to the major histocompatibility complex molecule of pigs, whether referred to as the primary or second major histocompatibility complex. When the second major histocompatibility complex molecule is removed, CD4+ T cells are unable to recognize genetically engineered endothelial cells; when the first and second major histocompatibility complex molecules are removed Except, the modified cells were not recognized by either CD4+ or CD8+ cells.

Preferred stem cell genetic modifications include 1) disruption of the endogenous non-mutant invariant chain gene, which acts on the assembly and delivery of a second class of MHC molecules to the cell surface, and loading of the antigenic peptide; and 2) disruption An endogenous β ₂ -microglobulin gene (β ₂ M gene) that encodes a protein required for all first-class MHC molecules to be expressed on the cell surface. In addition, only the non-mutant invariant chain gene is disrupted. A non-variant invariant chain is considered to be required for the insertion of an antigenic peptide fragment into a second class of MHC molecules. In summary, antigenic peptides and major histocompatibility complexes are recognized by T cells. When the antigen peptide is not present, T cell recognition cannot be achieved normally, and the second type of MHC molecule cannot be correctly folded. Therefore, in cells lacking a non-variant invariant chain, the presentation of the peptide will be hindered, and even if there is a very small amount of cell surface MHC, it still lacks the peptide and thus cannot cause an immune response.

The disruption of these genes can be achieved by homologous recombination gene targeting techniques. These techniques are conventional techniques. See U.S. Patent Nos. 6,916,654 and 6,986,887, Zijlstra et al., 1989, Nature 342:435438; and Koller et al., 1990 Science 248: 1227-1230.

9. Composition

The present invention provides a pharmaceutical composition comprising the aforementioned cells or active ingredients/compounds. The pharmaceutical compositions can be prepared by admixing a therapeutically effective amount of such cells or active agents/compounds, and optionally, other active materials, and a pharmaceutically acceptable carrier. The carrier can be in a variety of forms depending on the route of administration. Examples of other active substances include known active compounds or those identified by the aforementioned screening methods.

The aforementioned pharmaceutical compositions can be prepared by conventional pharmaceutical excipients and preparation methods. All excipients can be mixed with disintegrants, solvents, granulating agents, humectants and binders. The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount that results in a significant improvement in at least one symptom or parameter of a particular disease. The therapeutically effective amount of the aforementioned cells can be determined by conventional methods. An effective amount for treating a disease can be readily determined by a person of ordinary skill in the art using conventional test methods. The exact amount used to treat a patient will vary depending on the condition and severity of the condition and the physical condition of the patient. Significant improvement in any of the symptoms or parameters can be determined by the skilled artisan or by the physician's report of the patient. It will be appreciated that any clinical or statistically significant reduction or improvement of any of the symptoms or parameters of the aforementioned disorders is within the scope of the invention. A clinically significant decrease or improvement is meant to be apparent to the patient and/or to a physician.

The phrase "pharmaceutically acceptable" means that when administered to a human, the individual molecules and other components of the compositions are physiologically acceptable and generally do not produce undesirable reactions. Preferably, the term "pharmaceutically acceptable" means to be recognized by a federal regulatory agency, or a state government, or listed in the U.S. Pharmacopoeia, or other generally recognized pharmacopeia for use in mammals, particularly humans. Pharmaceutically acceptable salts, esters, guanamines, and prodrugs, which refer to such salts (eg, carboxylates, amino acid addition salts), esters, guanamines, and prodrugs, for reliable medical care Within the scope of judgment, it may be suitable for use in contact with patient tissues without transitional toxicity, irritation, allergic reactions, etc.; and the reasonable benefit/risk ratio and effectiveness of such uses need to be considered.

A carrier which can be applied to the aforementioned pharmaceutical composition means a diluent, an excipient, or a carrier which can be administered a compound. The pharmaceutical carriers can be sterile liquids such as water and oil. Preferably, water or an aqueous solution, a saline solution, and a liquid glucose and glycerin solution are used as a carrier, particularly as an injection solution. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", 18th edition, by E. W. Martin.

The aforementioned cells can be administered by injection or injection (for example, intravenous, intrathecal, intramuscular, intraluminal, intratracheal, intraperitoneal or subcutaneous), oral, skin penetration, or other conventional methods for administration to a body. . The dose may be administered once every two weeks, once a week, or more frequently, but when the disease or disorder is in a stable phase, the frequency of administration may be reduced.

Both heterologous and autologous cells can be used. In the former case, an HLA match will avoid or reduce the host's response. In the latter case, the autologous cell line is enriched and purified from a patient and stored for later use. The cells can be cultured in vitro in an environment containing the host, or transplanted T cells, and returned to the host. This allows the cells to be considered autologous in the host and to reduce T cell activity.

The dosage and frequency of administration will depend on the clinical symptoms, which are confirmed by the maintenance of the mitigation phase, or at least one or more of the acute phases, preferably more than one of the skilled practitioners of the field are known to have reduced clinical symptoms or disappear. More generally, the dose and frequency of administration will depend, in part, on the pathological condition of a disease condition or disorder and the impaired condition of the clinical and subclinical symptoms following treatment with the aforementioned compositions. The diagnosis and dosage of the patient or the treated breast-feeding patient and the physician can be adjusted according to age, gender, management of the physical condition, and conjugate benefits and side effects, all of which are skilled in the art. Know the person. In all of the foregoing methods, the cells are administered to a patient in an amount of from 1 x ^{10 4} to 1 x ^{10 10} each.

The following specific examples are illustrative only and are not intended to limit the disclosure of the invention. Without further elaboration, the skilled artisan skilled in the art can use the present invention to the fullest extent based on the description herein.

Preparation of SBR and SBT cells

Methods for activating, purifying, and amplifying BLSCs are described in WO/2007/100845. In this example, BLSCs were purified from the blood of a human patient in two ways. The isolated cell lines were analyzed by flow cytometry. The results are shown in Figures 1A to 1C and Figures 2A to 2C. As a result, it was found that the two methods obtained 200× ^{10 6} and 230× ^{10 6} BLSC/ml blood, respectively.

The purified BLSCs can be cultured in StemBios-001. To generate SBR and SBT cells, BLSCs can be cultured in StemBios-002 medium for two weeks. Next, the BLSCs are cultured in StemBios-003 medium containing 0.1 to 20 μM RA, or 1 to 40 nM TGF-β for more than two to six weeks. It can be observed that the morphology and size of the cultured cells gradually change (Figs. 6 to 12). These cells were named SBR and SBT cells, respectively.

The SBR and SBT cell lines were tested for developmental and differentiation capacity by standard methods. These cells can be found to differentiate into cells derived from three germ layers, such as ectoderm, mesoderm, and endoderm. It can also be observed that all or most of these cells are CD10 ⁺ , CD90 ⁺ , CD105 ⁺ , and CXCR4 ⁺ .

RT-PCR results of SBR, SBT and BLSCs

The gene expression of SBR cells, SBT cells and BLSCs can be detected. Total RNA was isolated from SBR cultured cells or SBT cultured cells (more than six million each) using a Qiagen RNA extraction kit (Qiagen, CA), Paris kit (Ambion), or RNAzol kit (InvitroGen). 2.0 μg of RNA can be prepared as cDNA using 0.25 ng of oligo-(dT) 12-18 and reverse transcriptase (Qiagen) according to the manual (Qiagen). However, detectable RNA is not available from more than 100 million BLSCs.

The RT-PCR was carried out for 30 cycles at 94 ° C (15 seconds), 55 ° C (15 seconds), and 72 ° C (20 seconds). The primer pair of β-actin is: forward 5'-ACAAAACCTAACTTGCGCAG-3'; reverse 5'-TCCTGTAACAACGCATCTCA-3'. The primer pair of GAPDH is: forward 5'-AGCCACATCGCTCAGACACC-3'; reverse 5'-GTACTCAGCGGCCAGCATCG-3'. These results are shown in Figure 13. It can be found that β-actin and GAPDH can be expressed in SBR cells and SBT cells, but not in BLSCs.

Other specific embodiments

All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by another alternative, equivalent, or similar. Therefore, unless expressly stated otherwise, the features of each disclosure are merely examples of the general series of various equivalent or similar features.

In the foregoing description, those skilled in the art can readily recognize the necessary technical features of the present invention, and various changes and modifications can be made to the various uses and conditions without departing from the spirit and scope of the invention. . Accordingly, other specific embodiments are also within the scope of the following claims.

1A to 1C show by hemolysis (hemolysis) to obtain mesodermal cells - similar to the stem cell population (BLSCs are) the flow cytometry histograms (flow cytometry histograms) of the result (the number of 200 × 10 ⁶ cells / ml).

2A to 2C show the results of flow cytometric histograms of embryonic leaf cell-like stem cell populations (BLSCs) obtained from plasma fractions (quantity 239×10 ⁶ cells/ml).

The third to third C-pictures are photographs showing that the cultured embryonic leaf-like stem cell populations (BLSCs) form a multi-layer, mesh net structure (200x).

Figure 4 is a photograph showing cultured embryonic leaf cell-like stem cell populations (BLSCs) forming aggregates.

Figure 5 is a photograph showing that cultured embryonic leaf cell-like stem cell populations (BLSCs) form spheroid cell aggregates.

Figures 6 to 8 are photographs showing the preparation of SBR cells from culture of embryonic leaf cell-like stem cell populations (BLSCs) containing retinoic acid.

Figures 9 to 12 are photographs showing the preparation of SBT cells from culture of embryonic leaf cell-like stem cell populations (BLSCs) containing transforming growth factor beta (TGF-β).

Figure 13 is a photograph of the results of RT-PCR showing the performance of GAPDH or β-actin in SBR and SBT cells, and not in BLSC. First: 1Kb mark. Second: 100bp mark. Third: SBR RNA for beta actin primers. Fourth: SBT RNA for beta actin primers. Fifth: for embryonic leaf cell-like stem cell (BLSC) RNA of beta actin primer. Sixth: no RNA, only beta actin primer. Seventh: SBR RNA for GAPDH primers. Eighth: SBT RNA for GAPDH primers. Ninth: Embryonic cell-like stem cell (BLSC) RNA for GAPDH primers. The tenth: no RNA, only GAPDH primer.

Claims (32)

A method of producing a pluripotent or pluripotent cell population comprising: obtaining a plurality of embryonic leaf cell-like stem cells (BLSCs) in a medium containing vitamin A (RA) or transforming growth factor beta (TGF-β) The BLSCs are cultured, and the pluripotent or totipotent cells in the cultured cells are identified and enriched; wherein the pluripotent or totipotent cells exhibit mRNA of GAPDH or β-actin. The method of claim 1, wherein the pluripotent or totipotent cells are 1 to 15 microns in size. The method of claim 1, wherein the medium contains 0.1 to 20 μM RA and the BLSCs are cultured for 2 to 8 weeks. The method of claim 3, wherein the medium contains 1 to 15 μM RA and the BLSCs are cultured for 2 to 8 weeks. The method of claim 4, wherein the medium contains 5 to 12 μM RA and the BLSCs are cultured for 3 to 4 weeks. The method of claim 5, wherein the pluripotent or totipotent cells are 1 to 15 microns in size and form a sheet-like structure in the suspension. The method of claim 1, wherein the medium contains 1 to 40 nM TGF-β and the BLSCs are cultured for 2 to 8 weeks. The method of claim 7, wherein the medium contains 2 to 20 nM TGF-β and the BLSCs are cultured for 2 to 8 weeks. The method of claim 8, wherein the medium contains 5 to 12 nM TGF-β and the BLSCs are cultured for 4 to 6 weeks. The method of claim 9, wherein the pluripotent or totipotent cells are 1 to 15 microns in size, have a circular shape and form agglomerates. A composition comprising a plurality of cultured cells, wherein the cultured cells (1) are pluripotent or pluripotent, and (2) are 1 to 15 microns in size, And (3) expressing mRNA of GAPDH or β-actin, wherein the cultured cells are prepared by obtaining a plurality of embryonic leaf cells-like stem cells (BLSCs) containing vitamin A acid (RA) or transgenic BLSCs were cultured in a medium of growth factor beta (TGF-β), and cells having the characteristics of (1), (2), and (3) in the cultured BLSC cells were identified and enriched. The composition of claim 11, wherein the composition further comprises vitamin A acid (RA) or transforming growth factor beta (TGF-β). The composition of claim 11, wherein the first group of cells is CD66e ⁺ . The composition of claim 11, wherein the cells comprise a recombinant nucleic acid. The composition of claim 14, wherein the recombinant nucleic acid encodes a polypeptide and the cells comprise an mRNA encoding the polypeptide. The patentable scope of application of the composition according to item 11, wherein the cells of the second group is CD66e ^-. The composition of claim 11, wherein the cells do not exhibit a β2-microglobulin gene. The composition of claim 11, wherein the cells do not exhibit one or more proteins encoded by a major class I histocompatibility complex (MHC) gene that stimulates T lymphocyte mediators. Anti-cell reaction. The composition of claim 11, wherein the cells are CD10 ⁺ , CD90 ⁺ , CD105 ⁺ and CXCR4 ⁺ . The composition of claim 11, wherein the cells are negative for trypan blue staining. A use of a composition according to claim 11 in the preparation of a medicament for treating a degenerative disease in a patient. The use of claim 21, wherein each cell in the composition comprises a recombinant nucleic acid. The use of claim 22, wherein the recombinant nucleic acid encodes a polypeptide and the cell comprises an mRNA encoding the polypeptide. The use of claim 23, wherein the degenerative disease is diabetes, neurodegenerative disease, arthritis or cancer. The use of claim 24, wherein the neurodegenerative disease is Parkinson's disease. A use of a composition according to claim 11 in the preparation of a medicament for treating an autoimmune disease in a patient. A method for identifying a drug candidate for treating a degenerative disease, the method comprising: contacting a test compound with a composition as claimed in claim 11 and determining a performance amount of a polypeptide which is down-regulated in a degenerative disease, wherein If the amount of expression in the presence of the test compound is higher than the amount of expression in the absence of the test compound, it indicates that the compound is a candidate for the treatment of the disease. The method of claim 27, wherein the degenerative disease is diabetes, a neurodegenerative disease, arthritis, cancer or an autoimmune disease. A cell bank comprising a plurality of compositions as in claim 11 of the patent application. For example, the cell bank of claim 29, wherein the cells are human cells. A method of manufacturing a cell bank according to claim 29, comprising: harvesting cells from a plurality of patients to obtain a plurality of embryonic leaf cell-similar stem cell populations respectively; identifying the cell populations, obtaining at least one for each cell population Scheduled Characterizing; and classifying each of the groups of cells in accordance with the at least one predetermined characteristic. The method of claim 31, wherein the method further comprises expanding the population of cells.