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WO2001027266A1 - Segments d'adn utilises pour la selection de types cellulaires differencies, et applications correspondantes - Google Patents

Segments d'adn utilises pour la selection de types cellulaires differencies, et applications correspondantes Download PDF

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Publication number
WO2001027266A1
WO2001027266A1 PCT/ES2000/000391 ES0000391W WO0127266A1 WO 2001027266 A1 WO2001027266 A1 WO 2001027266A1 ES 0000391 W ES0000391 W ES 0000391W WO 0127266 A1 WO0127266 A1 WO 0127266A1
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gene
cells
cell
cell type
dna segment
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PCT/ES2000/000391
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English (en)
Spanish (es)
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Alfonso Valera Abril
Josefa María MAYOL BELDA
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Universidad Miguel Hernandez De Elche
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Priority to AU77913/00A priority Critical patent/AU7791300A/en
Publication of WO2001027266A1 publication Critical patent/WO2001027266A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays

Definitions

  • the invention relates, in general, to obtaining a differentiated cell type from precursor cells, and, in particular, to DNA segments useful for the selection of differentiated cell types and their applications, among those found, obtaining and in vitro selection of differentiated cell types from precursor cells. With the cells thus obtained, cell and tissue banks can be generated for biomedical, biotechnological or research purposes.
  • Diabetes mellitus is a serious metabolic disease with a huge socioeconomic impact, which affects between 10 and 20 million people in the European Union (about 5% of the population) [Hauner et al., 1992; de Leiva et al., 1996 (see the section on Bibliography)] and with an estimated worldwide incidence of around 6% [Taylor et al., (1988); McGarry, (1992)]. All forms of diabetes are characterized by hyperglycemia derived from partial or total insulin deficiency, with the long-term appearance of alterations in the glomerulous area of the kidney and neurological complications [illiamson et al., (1993); Ruderman et al., (1992); Diabetes Control and Complications Trial Research Group, (1993)].
  • IDDM Insulin-dependent diabetes mellitus
  • type 1 involves the destruction of insulin-producing beta cells from the islets of Langerhans in the pancreas.
  • the destruction of beta cells in type 1 diabetes is considered the result of an autoimmune attack, in which the patient's own immune system recognizes and destroys their beta cells, although not the rest of the cell types that complete the islet [Eisenbarth, (1986)].
  • type 1 patients suffer episodes of abnormally high blood glucose levels (hyperglycemia) [Nathan, (1992)]. These uncontrolled and recurrent episodes of hyperglycemia are considered the cause of the development of secondary complications that lead to renal failure, blindness, neuropathy or cardiovascular diseases [Nathan, (1992)].
  • the maintenance of blood glucose under strict control is widely recognized as crucial in the treatment of all forms of diabetes to delay the onset and reduce the severity of microvascular complications and some macrovascular complications.
  • beta cell lines could be modified to carry out the synthesis and secretion of insulin regulated by nutrients.
  • hamster beta cells could be modified to carry out the synthesis and secretion of insulin regulated by nutrients.
  • hamster beta cells could be modified to carry out the synthesis and secretion of insulin regulated by nutrients.
  • hamster beta cells could be modified to carry out the synthesis and secretion of insulin regulated by nutrients.
  • hamster beta cells could be modified to carry out the synthesis and secretion of insulin regulated by nutrients.
  • pluripotential stem cells to obtain cells and tissues for transplantation.
  • Success in the cultivation of human pluripotential embryonic cells [Gearhart, (1998)] opened high expectations for the treatment of neurodegenerative diseases, diabetes, spinal cord injury, hematopoietic repopulation, or repair and regeneration of damaged tissue.
  • these results allowed us to think about the medium-term creation of stem cell cell banks, the creation of universal donor cell lines, or even make embryonic cells of an adult individual through nucleus transfer.
  • the conditions for inducing directed differentiation of stem cells are yet to be defined.
  • Today, in vitro differentiation studies of embryonic pluripotential cells are based on the selection and / or enrichment of specific cell lineages among all that may exist.
  • Ott et al. (1994) have used a method that relies on mechanisms in vivo to obtain differentiated specific cell lineages from an embryonic pluripotential cell.
  • This is basically a method for the characterization of cell lineages derived from neuronal stem cells with a fragment of marker DNA that were reintroduced into embryos in early stages of development.
  • the marker gene is expressed during neurogenesis and the cells that express it are dissected and maintained in culture. This method allows the histological identification and isolation of determined cells towards the neuronal lineage that continues until terminal differentiation in culture.
  • Gay US Patent 5,639,618 (1997) proposes to use a marker gene whose expression can be followed by a fluorescence activated cell separation system (FACS) or by immuno-cytochemistry.
  • FACS fluorescence activated cell separation system
  • the method preferably uses membrane-expressed marker proteins
  • the method is restricted to the Otx, Dlx, Nlx, Emx, Wnt, En, Hox, and ACHR13 genes, which are genes that code for large groups of cell lineages and are not specific to a specific cell type.
  • Transference is an irreversible process whereby a differentiated cell becomes a different cell type. There is much confusion in the terminology used to indicate changes in cell differentiation.
  • Metaplasia is the traditional term used to indicate the change from a differentiated cell type to another cell type. This term was introduced based on anatomical and histological observations of tissues of Unexpected and ectopic appearance. Metaplasia may appear as a result of selective growth of very minor cells present in a particular organ. In other cases, the new differentiation may occur as a result of the re-differentiation of pre-existing differentiated cells.
  • Transdetermination denotes an alteration in the future differentiation pathways of a previously determined but not yet differentiated cell. From a traditional point of view, all developing cells must go through a state of determination before differentiation.
  • Differentiation means the process by which a cell loses the differentiated state and expresses an embryonic phenotype. Dedifferentiation can be applied to any change to a new state in which the cell loses its initial characteristics before the appearance of a new specificity.
  • Modulation is another term used to indicate flexibility in the state of differentiation. There are numerous examples described in the literature [Okada, 1991].
  • An object of this invention is a segment of DNA, useful for selecting distinct distinct cell types, comprising (i) the regions that contribute to form and stabilize the complex of transcription factors of a specific gene of a cell type that is expressed specifically in a differentiated cell type, and (ii) a functionally connected selection gene, so that it is expressed when the DNA segment is recognized and transcribed by the transcription factor complex of the specific gene.
  • a further object of this invention is a method for obtaining a differentiated cell type comprising the use of said segment of DNA to transfect precursor cells of said type. differentiated cell.
  • a method for maturing in vitro until differentiation obtained differentiated cells also constitute an object of this invention.
  • the precursor cells transiected with said DNA segment as well as the differentiated cells obtained from said transcribed precursor cells constitute additional objects of the present invention.
  • Another additional objects of the present invention are methods for obtaining stem cells from adult mammalian individuals, as well as certain stem cells, which comprise the use of said DNA segment.
  • Figure 1 is a schematic representation of the DNA segment used in the embodiment of Example 1.
  • the arrows indicate the approximate cut sites of the corresponding restriction enzymes.
  • Figure 2 is a graph depicting the level of blood glucose at different times in transplanted animals and in controls [see Example 1].
  • Figure 3 is a graph showing the evolution of body weight over time in transplanted animals and in controls [see Example 1].
  • Figure 4 is a graph representing the results of the glucose tolerance test at different times in transplanted animals and in controls [see Example 1].
  • Figure 5 are photomicrographs of a histological section of the spleen of a representative transplanted animal, analyzed by immunohistochemical techniques that reveal the presence of insulin as ocher staining [see Example 1].
  • Figure 5A shows a diabetic pancreas section without islets and
  • Figure 5B shows a transplanted spleen section.
  • Figure 6 are photomicrographs of histological sections of the tissues derived from the transplanted lines in the immunosuppressed mice [see Example 3], specifically, of mesodermal tissue (Figure 6A), of endodermal and mesodermal tissues ( Figure 6B), and of tissues Endodermal, ectodermal and neuro-ectodermal ( Figure 6C).
  • the DNA segment Transcriptional control is crucial for cell differentiation since it determines which genes are transcribed and therefore what proteins the cell will produce. Gene expression is regulated by the coordinated action of regulatory proteins (transcription factors) that bind to key regions of the DNA to control gene expression.
  • regulatory proteins transcription factors
  • the interactions of the transcription factors can be very complex and the number, type and combinations of the regulatory proteins that form the transcription complex is known only very partially and in a few cases. Therefore, the possibility of directing Cellular differentiation using this little knowledge is excessively limited.
  • each gene is activated by a specific and unique combination of factors that, as a whole, recognize and transcribe the DNA of that gene among the tens of thousands of genes present in The genome of a mammalian cell. Therefore, if the DNA segment that is recognized by the transcription complex is supplied, any gene (for example, a selection gene) can be functionally inserted into that DNA segment only when the combination of factors that form the complex of DNA segment transcription found in the cell.
  • any gene for example, a selection gene
  • the key point is to construct a segment of DNA that is recognized by the transcription complex of a specific gene of a particular cell type and that allows to select said cell type.
  • the segment of DNA that allows successful selection of pancreatic beta cells from mouse pluripotential cells, human teratocarcinoma cells, and murine and human adult intestinal cells should be recognized by the transcription complex of the gene of insulin.
  • the specific gene used must be of the species to which the precursor cells belong.
  • Transcription factor means any protein required to initiate or regulate transcription. It includes both gene group specific regulatory proteins and common proteins of the general transcription machinery.
  • the "transcription factor complex” is the group of factors that recognizes the regulatory regions of a gene's DNA to initiate its transcription.
  • the "DNA regulatory regions” include not only the binding sequences for transcription factors, identified or not, but also other regions that have a relevant role in the DNA binding of the transcription factor complex.
  • segment of DNA is meant, in general, any exogenous DNA that contains a selection gene functionally connected to the regulatory regions of DNA of a specific gene. This concept is especially relevant in the development of the present invention.
  • This fragment of DNA regions called the DNA segment is defined exclusively by the final result of the selection of the specific cell type with a defined phenotype.
  • the invention provides a DNA segment, useful for selecting differentiated cell types, sometimes called the DNA segment of the invention, comprising
  • the specific gene is a gene that is preferably expressed in a single differentiated cell type, although specific genes that are expressed in more than one differentiated cell type can also be used although they preferably do so in a specific differentiated cell type. Therefore, in general, any specific gene of a cell type that is specifically expressed in a differentiated cell type can be used in the DNA segment of the invention.
  • said specific gene of a cell type that is expressed specifically in a differentiated cell type is a selected mammalian gene, including man.
  • the insulin gene would be used to obtain pancreatic beta cells, the rhodopsin gene for retinal photoreceptors, the ornithine transcarbamylase gene for the liver or the albumin gene, these being understood as non-limiting examples of specific genes of differentiated cell types.
  • the selection gene is a gene whose expression provides a phenotypic characteristic that allows selecting cells transfected with said selection gene. Therefore, any selection gene, which meets the previously established condition, can be used in the DNA segment of the invention.
  • said selection gene is selected from a resistance gene, for example, the geneticin resistance gene, the dihydrofolate resistance gene, the puromycin resistance gene, the interferon gamma resistance gene, and the methotrexate resistance gene (multidrug resistance gene), a temperature gene and its combinations.
  • a strategy to increase the specificity of the procedure for the selection of said cell type involves the use of a secondary gene. which is also expressed in the specific cell type. After the insertion of another positive selection gene, different from the one that is functionally connected to the specific gene, in a region of the secondary gene that allows the expression of the second selection gene when the DNA regions of the secondary gene are recognized by their complex of transcription factors, specific genes are introduced and secondary, and their respective selection genes, in the precursor cell to differentiate. In this case, cell type selection could be done by double positive selection.
  • the secondary gene can be expressed in other cell types, but the combination of the expression of the specific gene and the secondary gene must be exclusive to the phenotype of the cell type that is intended to be obtained.
  • the invention also provides a DNA segment of the invention, further comprising a secondary gene that is expressed in the cell type in which the specific gene is expressed, functionally connected to a second selection gene, other than selection gene connected to the specific gene.
  • said DNA segment further comprises the region that contributes to forming and stabilizing the complex of transcription factors of said secondary gene of the cell type to be selected.
  • any secondary gene of a cellular type in which a specific gene is specifically expressed can be used in the DNA segment of the invention as long as the combination of the expression of the specific gene and the secondary gene is exclusive to the phenotype of the cellular type that It is intended to be obtained.
  • said secondary gene is selected from the group consisting of the glucokinase gene or the Glut-2 glucose transporter gene for pancreatic beta cells and the phosphoenolpyruvate carboxykinase gene or the liver albumin gene .
  • the invention also contemplates the development of a DNA segment of the invention which further comprises 2 or more non-specific genes of the cell type to be selected but whose combined expression is specific to the cell type to be selected, operatively connected with their corresponding regulatory regions, and each of said regulatory regions being functionally connected to, at less, a selection gene.
  • the DNA segment of the invention may also advantageously contain a biological safety system that prevents contamination of cells with other unwanted cell types, and, preferably, an additional cellular biosecurity system, for example, the gene of the thymidine kinase from herpesvirus that confers sensitivity to ganciclovir, in order to pharmacologically remove, in vitro or in vivo, the manipulated cells.
  • an additional cellular biosecurity system for example, the gene of the thymidine kinase from herpesvirus that confers sensitivity to ganciclovir, in order to pharmacologically remove, in vitro or in vivo, the manipulated cells.
  • the invention provides a method for obtaining and isolating specific differentiated cells from precursor cells comprising the use of the DNA segment of the invention. More specifically, the invention provides a method for obtaining in vitro a differentiated cell type from precursor cells, sometimes referred to as the method of the invention or Cell Type Selection Procedure, which comprises the general steps of: a ) transfect precursor cells with a DNA segment of the invention; b) culturing the transfected precursor cells under suitable conditions so that they become the type differentiated cell; c) select the differentiated cell type that expresses the specific gene and, consequently, the selection gene; and d) cultivate and grow the differentiated cell type until reaching the required amount.
  • the precursor cells before being transfected with the DNA segment of the invention, are cultured under conditions that ensure that they are maintained in an undifferentiated state, for example, by addition to the culture medium [such as Dulbecco's modified essential medium
  • DMEM fetal bovine serum
  • factors that inhibit spontaneous differentiation of cells such as the leukemia inhibitory factor (1,000 U / ml, ESGRO, Life Technologies, Inc.).
  • Undifferentiated precursor cells in a number of approximately 10 5 -10 7 cells per transfection session, are transfected with a DNA segment of the invention.
  • the suitable conditions for culturing the transfected precursor cells so that they become the differentiated cell type focus on creating a suitable in vitro microenvironment so that differentiation gradients can be established in aggregate cell groups
  • embryonic bodies so that certain cells appear towards multiple cell lineages; for this, the precursor cells are grown, for example, in DMEM with 10% fetal bovine serum, the factors that inhibit differentiation are removed, and are maintained in suspension culture, so that the cells form spontaneously aggregates and after 2-7 days these become embryonic bodies [Robertson, (1987)].
  • the embryonic bodies are placed in culture plates to which they can adhere and the differentiated cell type that expresses the gene is selected specific, and, consequently, the selection gene, by modifying the culture conditions (for example, by adding a cytotoxic agent for 1-2 weeks) that will eliminate any cell that does not have the specific gene activated, from this mode only the cell type of interest will survive and phenotypically homogeneous cultures will be available in terms of specific gene expression.
  • the differentiated cell type selected is cultivated and grows, for example, culture conditions adherent (monolayer culture plate) up to the amount needed (usually 10 4 to 10 to 10 cells) for therapeutic response effective for research trials, to produce biotechnological derivatives, or for any other type of application.
  • culture conditions adherent monolayer culture plate
  • terminal differentiation can be initiated, in the case that the cell type sought has that characteristic (for example, in the case of obtaining pancreatic beta cells, neurons, cardiomyocytes, corneal epithelial cells, blood cells, chromaffin cells of the adrenal medulla or photoreceptors of the retina).
  • the conditions for initiating terminal differentiation will vary depending on the cell type, for example, adding differentiating factors to the culture medium; thus, retinoic acid and cyclic adenosine monophosphate (cyclic AMP) will be added to obtain neurons; dimethylsulfoxide (DMSO) will be added to obtain cardiomyocytes; glucocorticoids will be added to obtain chromaffin cells; or nicotinamide will be added to obtain pancreatic beta cells.
  • DMSO dimethylsulfoxide
  • glucocorticoids will be added to obtain chromaffin cells
  • nicotinamide will be added to obtain pancreatic beta cells.
  • the invention provides a method for obtaining precursor cells of adult human individuals from tissue samples that retain the ability to multiply in vivo, such as the epithelium.
  • tissue samples that retain the ability to multiply in vivo, such as the epithelium.
  • Example 3 a protocol similar to that detailed in Example 3 (applied to epithelial cells) is followed, which briefly comprises the gentle disintegration of the tissue sample by using a buffered solution containing a reducing agent, for example dithiothreitol, and a calcium chelating agent, for example, ethylenediaminetetraacetate (EDTA), followed by culture for several days (1-3 weeks) under conditions that keep the disintegrated cells in undifferentiated state
  • a reducing agent for example dithiothreitol
  • a calcium chelating agent for example, ethylenediaminetetraacetate (EDTA)
  • any of the more than 200 cell types can be obtained differentiated that make up the human body (as an example, see the cell types described above), as well as precursor cells of adult individuals that are determined to give rise to a specific lineage.
  • red series precursor cells monocyte precursors, neutrophil precursors, or lymphocyte precursors
  • glial cells astrocyte precursors, oligodendrocytes, schwann cells, ependymal cells or microglia
  • precursors of the different specialized types of neurons present in the seven anatomical regions of the central nervous system or in the case of the cells that make up the pancreatic islets, precursor cells of specialized endocrine types (alpha cells or producers of glucagon, beta or insulin producers, delta or somatostatin producers, or PP cells).
  • the method of the invention allows differentiated cells to be created with the immunological characteristics of the individual himself. Such personal cells would be suitable for eventual transplantation to the individual himself and his use would avoid the problem of immune rejection in case of autotransplantation.
  • the process of the invention opens the possibility of obtaining biological material from the individual himself for the effective cell therapy of many diseases, virtually any somatic disease, for example, diabetes mellitus, Parkinson's disease, retinitis pigmentosa, etc.
  • the method of the invention is a continuous source of cells of a certain selected type, which allows new therapeutic approaches for transplants, for example, liver transplantation or pancreatic, the replacement of muscle cells in the case of muscular dystrophy or myocardial infarction, the replacement of blood cells in cases of cancer in the hematopoietic tissue, etc.
  • the process of the invention is illustrated below with specific examples of obtaining pancreatic beta cells that can be used in multiple applications, for example, in the treatment of diabetes mellitus (both type 1 and type 2), in the detection of antigens associated with diabetes, and even in the large-scale production of properly packaged insulin.
  • the method of the invention is based, among other things, on that under controlled in vitro culture conditions both embryonic cells and adult individual cells can differentiate other cell types.
  • the process of the invention allows (i) to obtain cell lines with which to obtain, select and isolate cell types as poorly represented in the culture as 1 cell among 10 8 cells, and (ii) the expansion and maturation of the selected cells until obtaining the specific cell type of therapeutic utility.
  • Precursor cells One of the greatest difficulties in defining the precursor cells is that they are if they have certain functional capabilities, which can only be verified by checking that they really do. Initially, a population of precursor cells that had a great capacity to self-maintenance [Lajtha, (1970)]. A more recent definition distinguishes between active, potential, lineage-specific and determined stem cells [Potten and Loeffler, (1990); Potten, (1996)]. "Active stem cells” are undifferentiated precursor cells capable of proliferating, self-sustaining, producing a large number of derived cell lineages, regenerating tissues after injury, and possessing flexibility in the use of those capacities. “Potential stem cells” are the latent or quiescent version of active stem cells.
  • Determined stem cells are transient cells with the ability to divide and that can share with the terminal cells differentiated the ability to perform specific tissue functions. Some of these specific stem cells give rise to a few cell types, and in many cases they are in fact unipotential precursor cells, determined to a single cell type. In this sense it is different from the "specific stem cell of lineage", which is a precursor cell determined to give rise to all cell types of a certain lineage, for example the neuronal lineage, etc.
  • “Adult stem cells” refers to a population of precursor cells that are capable of dividing and terminal differentiation only after they have multiplied. These adult stem cells are responsible and necessary for the growth of tissues during development, the regeneration of certain organs such as the liver, or the continuous production of blood or skin cells. The results obtained by the inventors indicate that, under certain conditions, adult precursor cells can also be pluripotential.
  • “Pluripotential stem cell” a term that has so far been restricted to embryonic stem cells, refers to those precursor cells that can differentiate a wide variety of cell lineages.
  • the “embryonic stem cell,” which is also pluripotential, is one that has been isolated from an embryo.
  • the "immortal stem cell” is defined as a cell population that permanently retains its embryonic characteristics and that can multiply in culture unlimitedly, or differentiate multiple lineages and cell types under certain conditions. According to the results of the inventors, the origin of these immortal stem cells may be an adult stem cell.
  • Any precursor cell can be used for carrying out the process of the invention, for example, pluripotential embryonic stem cells, embryonic carcinoma cells, or cells of adult human individuals who, under defined culture conditions, behave as stem cells (adult stem cells), for example, gastrointestinal epithelial cells, skin cells, ocular limbus epithelial cells, mammary gland cells, liver cells, mucosal epithelial cells, hematopoietic cells, ependymocytes, or cells pancreatic ductals.
  • stem cells adult stem cells
  • adult stem cells for example, gastrointestinal epithelial cells, skin cells, ocular limbus epithelial cells, mammary gland cells, liver cells, mucosal epithelial cells, hematopoietic cells, ependymocytes, or cells pancreatic ductals.
  • Example 3 The results obtained by the inventors [see Example 3] show that cell lines generated from adult mammals (mouse and human) have the potential to give rise to cell types and tissues (neuronal tissue, mucosecretory epithelium, striated muscle, cartilage , bone, etc.) derived from the three embryonic layers (ectoderm, mesoderm and endoderm).
  • transfection refers to any method of introducing a segment of DNA into a eukaryotic cell.
  • the term thus defined includes both methods that employ viral systems (for example, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, Sindbis viruses, etc.) and non-viral (electroporation, lipofection, calcium phosphate mediated, by DEAE dextran, by cellular uptake, by mechanical methods, etc.), and also by any other method of DNA transfer
  • the selection of the cell type from embryonic or adult stem cells allows selected stem cells to be pancreatic beta cells.
  • the selection of the differentiated cell type that expresses the specific gene is carried out in culture.
  • the selection of said differentiated cell type that expresses the specific gene can be carried out by a double positive selection method using a DNA segment of the invention comprising a secondary gene, functionally connected to another selection gene different from that connected to the specific gene. , in addition to the specific gene.
  • pancreatic beta cell When the specific gene corresponds to a terminal differentiated cell, the selected cell must be a determined stem cell that maintains its ability to divide until other factors cause its terminal differentiation. In the case of the pancreatic beta cell, there are numerous extracellular factors that inhibit its proliferation [Hügl et al., (1998)]. The most effective are inhibitors of phosphoproteinophosphatases, such as orthovanadate, or inhibitors of intracellular signals mediated by Protein kinases that phosphorylate tyrosine residues, such as genistein, wortmanin, and compound 2- (4-morpholinyl) - 8-phenyl-4H-l-benzopyran-4-one identified as LY294002 [Vlahos et al.
  • betacellulin The process that leads to the maturation of the beta cell is not well known and is an active field of research. This process involves both cell proliferation and terminal differentiation. Since pancreatic beta cells do not divide, this process requires opposite behaviors in practice. Some factors such as betacellulin, tumor growth factor alpha, nerve growth factor, or activin A activate proliferation, while nicotinamide, sodium butyrate, vitamin D 3 or genistein and wortmanin inhibitors stop proliferation. .
  • nicotinamide causes growth and maturation of pancreatic beta cells [Otonkoski et al., (1993); Anderson et al., (1994)], and hepatocytes [Mitaka et al., (1995); Fardel et al., (1992)].
  • the combination of vitamin D 3 and sodium butyrate has been described as very effective in inhibiting the growth of pancreatic beta cells [Lee et al., (1994)].
  • Example 1 that accompanies this description demonstrates that the determined stem cells obtained after the selection of cells expressing the specific gene were already beginning to manifest incipient phenotypic characteristics of pancreatic beta cells (insulin content, insulin release to the medium).
  • pancreatic beta cells insulin content, insulin release to the medium.
  • a differentiation culture medium with 5-25 mM nicotinamide followed by 1-3 weeks in another terminal differentiation culture medium with 3-5 mM glucose and 5-25 mM nicotinamide was generated a pure cell population that had an insulin content just like the mature pancreatic beta cell, which it responded efficiently and physiologically to the secretagogues just like the mature pancreatic beta cell, and that they did not divide, nor do mature beta cells.
  • the present invention is universal in nature, which includes obtaining any cell type of interest and its applications, biomedical, biotechnological or research, for example, obtaining cells for the treatment of diseases that occur with alteration, degeneration and / or cell loss (diabetes, Parkinson's, Alzheimer's, heart disease, retinitis pigmentosa and other retinopathies, leukemia, musculoskeletal disorders, liver disease, acquired immunodeficiency syndrome, non-regenerative anemias, etc.), obtaining products derived from specific cell types for the development of products useful in biomedical, biotechnological or research applications; for example, obtaining drugs of biological origin for blocking tumor dedifferentiation, or anti-idiotype antibodies for blocking undesirable immune reactions, obtaining cellular antigens for the detection of antibodies useful for the diagnosis of diseases; Obtaining biological reagents for research aimed at developing new diagnostic, prognostic or disease treatment procedures; use of the cells obtained by the method of the invention for the study of the processes that lead to differentiation and cell dedifferentiation
  • precursor cells transfected with a DNA segment of the invention as well as differentiated cells obtained from said precursor cells constitute additional objects of the present invention.
  • the invention provides a method for culturing in vitro differentiated cells in liquid medium, for example, pancreatic beta cells obtainable by the process of the invention, comprising culturing in vitro said differentiated cells obtainable by the process of the invention in a liquid medium under conditions that allow the growth of said differentiated cells.
  • the invention also provides a method for obtaining a product of interest comprising the expression of differentiated cells obtainable by the process of the invention.
  • said product of interest is selected from drugs of biological origin for blocking tumor dedifferentiation, anti-idiotype antibodies for blocking undesirable immune reactions, cellular antigens for the detection of antibodies useful for the diagnosis of diseases, and reagents Biological research aimed at developing new diagnostic, prognostic or disease treatment procedures.
  • said product of interest is insulin, and is obtained by peripheral pancreatic beta cells obtainable by the process of the invention in a buffered liquid medium containing secretagogues.
  • the invention provides a method for obtaining precursor cells from adult mammalian individuals, including the human species (see the application examples indicated above). These cells can be transfected with the DNA segment of the invention, cultured transfected cells under suitable conditions so that they become specific precursor cells of a particular cell destination and select said precursor cells that express the specific gene and the selection gene.
  • the invention provides a method for obtaining in vitro determinate precursor cells of adult mammalian individuals, including the human species, comprising transfecting cells of said adult mammalian individuals with the DNA segment of the invention, culturing the transfected cells under conditions suitable for become specific precursor cells and select said determined precursor cells that express the specific gene and the selection gene.
  • the invention also provides a method for maturing in vitro until terminal differentiation to the differentiated cell type obtained by the method of the invention comprising the addition of the factors necessary to achieve terminal differentiation of the differentiated cell type (see examples indicated above).
  • the invention also relates to the use of the DNA segment of the invention, of the differentiated cell types obtained by the method of the invention, optionally matured until terminal differentiation, of the precursor cells transfected with the DNA segment of the invention, in all type of applications, especially in therapeutic, biomedical or research applications (see the application examples indicated above).
  • the following examples demonstrate certain aspects of the implementation of the object of this invention. It is foreseeable that personnel with ordinary preparation in this field of research can use the invention to its full extent.
  • the examples presented below should be considered merely illustrative of the object of the present invention and, therefore, not limiting the scope thereof.
  • pancreatic beta cells obtained from mouse embryonic stem cells (or precursor cells)
  • CMEM murine embryonic stem cells
  • the segment of DNA whose characteristics are shown in Figure 1 was transfected to the CMEM by electroporation or lipofection, obtaining similar results in both cases, and culture of the transfected cells for 24-48 hours without any selection.
  • the DNA segment includes the regions that contribute to form and stabilize the complex of transcription factors of the human insulin gene (specific gene), a gene that confers resistance to the geneticin antibiotic (selection gene) functionally connected to the insulin gene human, a constitutive selection gene (hygromycin resistance gene) that allows the selection of cells that have incorporated the DNA segment and the thymidine kinase (TK) gene that confers sensitivity to ganciclovir (gain) (system of biosecurity).
  • the DNA segment was obtained by General molecular biology techniques, as indicated above.
  • transfected cells The selection of transfected cells was performed by adding to the hygromycin culture medium (0.1 mg / ml) for 10-14 days, and subsequent isolation and expansion of individual cell clones. All isolated clones gave similar results, and all of them differentiated mature pancreatic beta cells.
  • the selection of insulin-expressing cells from among differentiated cell clones in vitro was made by adding to the culture medium of the antibiotic geneticin (0.2 mg / ml) for 10-14 days, followed by cell expansion resistant appeared.
  • Cellular aggregates (containing 10 2 to 10 3 cells per aggregate) were generated following the protocol indicated in ld). After 2 days in culture, 20 mM nicotinamide was added to the culture medium and cell aggregates were cultured for 2 weeks. Then, the usual culture medium (modified minimum essential medium was changed) by Dulbecco, or DMEM) to the terminal differentiation medium consisting of a base of CMRL-1099 medium (Life Technologies) supplemented with 20 mM nicotinamide, and the cells were maintained in culture for another 7-14 days.
  • Table 1 shows the content and secretion of insulin depending on the evolution of the differentiation of several cell clones obtained by the Objective Cell Type Selection Procedure of the present invention.
  • pancreatic beta cells obtained from CMEM by the Cell Type Selection Procedure, the test described below was performed. Diabetic animals were made by a single intravenous injection of streptozotocin (0.2 mg / g live weight). Only extreme diabetic mice with blood glucose levels greater than 500 mg / dl were used for at least three days, and that had large amounts of glucose and ketone bodies in urine determined by urine test strip (+++) (Amestix , Ames). Differentiated cells (from section lf) in the form of cell aggregates (10 3 to 10 4 per mouse) were transplanted to the spleen of diabetic mice in a single session.
  • Example 2 shows the content and secretion of insulin before and after differentiation of several cell clones obtained by the Cell Type Selection Procedure object of the present invention.
  • pancreatic beta cells obtained from human teratocarcinoma cells by the Cell Type Selection Procedure Currently, the therapeutic evaluation phase is being carried out in vivo in immunosuppressed diabetic mice that are to be administered beta cells obtained by the Cell Type Selection Procedure provided by this invention.
  • EXAMPLE 3 Collection and evaluation of adult stem cells (or precursors) from a sample of human intestinal epithelial cells 3.
  • CMAH human adult stem cells
  • the establishment and culture of human adult stem cells (CMAH) obtained from a sample of human intestinal epithelial cells was carried out following the protocol described below: 1) incubation for one hour at 4 ° C of the sample in physiological saline to which 10 mM dithiothreitol was added;
  • isolation medium composed of DMEM standard medium supplemented with 15% heat-inactivated bovine fetal serum, a standard mixture of 100 mM non-essential amino acids (Life Technologies), 2- 0.1 mM mercaptoethanol, 10 micrograms / ml of streptomycin, 10 Units / ml of penicillin and 10 micrograms / ml of insulin;
  • EXAMPLE 4 Obtaining and therapeutic assessment of differentiated pancreatic beta cells from adult stem cells (or precursors) derived from a sample of murine intestinal epithelial cells
  • CMAM murine adult stem cells
  • CMAM murine adult stem cells
  • Example 1 The procedure described in sections lb) -lf) of Example 1 is also carried out, using multi-potential CMAM lines as cells to be transfected.
  • Table 3 shows the content and secretion of insulin before and after differentiation of several cell clones obtained by the Cell Type Selection Procedure object of the present invention.
  • Table 3 Insulin content and secretion depending on the differentiation of several independent cell lines obtained by the Cell Type Selection Procedure
  • the therapeutic evaluation phase is currently under way in vivo in diabetic mice to which the pancreatic beta cells obtained from CMAM are to be administered by the Cell Type Selection Procedure provided by the present invention.
  • EXAMPLE 5 Collection and therapeutic evaluation of human pancreatic beta cells differentiated from CMAH derived from a sample of human intestinal epithelial cells
  • Example 1 The procedure described in sections lb) -lf) of Example 1 is also carried out, using pluripotential CMAH lines as cells to be transfected.
  • Table 4 shows the content and secretion of insulin before and after differentiation of several cell clones obtained by the Cell Type Selection Procedure object of the present invention.
  • the therapeutic evaluation phase is currently under way in vivo in immunosuppressed diabetic mice to which human pancreatic beta cells obtained from CMAH are to be administered by the Cell Type Selection Procedure provided by this invention.

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Abstract

L'invention concerne un segment d'ADN comprenant (i) les régions qui contribuent à la formation et à la stabilisation du complexe des facteurs de transcription d'un gène spécifique d'un type cellulaire exprimé, de manière spécifique, dans un type cellulaire différencié, et (ii) un gène de sélection, fonctionnellement relié, de manière qu'il s'exprime lorsque le segment d'ADN est reconnu et transcrit par le complexe des facteurs de transcription du gène spécifique. Ledit segment d'ADN peut être utilisé pour l'obtention et la sélection in vitro de types cellulaires différenciés à partir de cellules précurseurs. Les types cellulaires obtenus permettent la génération de banques cellulaires et tissulaires à des fins thérapeutiques ou de recherche.
PCT/ES2000/000391 1999-10-14 2000-10-13 Segments d'adn utilises pour la selection de types cellulaires differencies, et applications correspondantes WO2001027266A1 (fr)

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AU77913/00A AU7791300A (en) 1999-10-14 2000-10-13 Dna segments that are useful for selecting differentiated cell types and the applications thereof

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ESP9902251 1999-10-14
ES9902251A ES2183665B1 (es) 1999-10-14 1999-10-14 Segmentos de adn utiles para seleccionar tipos celulares diferenciados y sus aplicaciones.

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994024274A1 (fr) * 1993-04-21 1994-10-27 The University Of Edinburgh Isolation, selection et propagation de cellules souches d'animaux transgeniques
WO1995020042A1 (fr) * 1994-01-25 1995-07-27 Ppl Therapeutics (Scotland) Limited Isolement de cellules souche embryonnaires
WO1998035022A1 (fr) * 1997-02-06 1998-08-13 Osiris Therapeutics, Inc. EFFETS DE LA p21CIP1 ET DE LA p27KIP1 SUR LA REGULATION DE LA DIFFERENCIATION DES CELLULES SOUCHES MESENCHYMATEUSES HUMAINES
WO1998044142A1 (fr) * 1997-04-01 1998-10-08 The General Hospital Corporation Marqueur moleculaire pour cellules souches musculaires
WO1999020740A2 (fr) * 1997-10-23 1999-04-29 Geron Corporation Procedes et matieres utiles pour la croissance de cellules souches primordiales de primate

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5753506A (en) * 1996-05-23 1998-05-19 Cns Stem Cell Technology, Inc. Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994024274A1 (fr) * 1993-04-21 1994-10-27 The University Of Edinburgh Isolation, selection et propagation de cellules souches d'animaux transgeniques
WO1995020042A1 (fr) * 1994-01-25 1995-07-27 Ppl Therapeutics (Scotland) Limited Isolement de cellules souche embryonnaires
WO1998035022A1 (fr) * 1997-02-06 1998-08-13 Osiris Therapeutics, Inc. EFFETS DE LA p21CIP1 ET DE LA p27KIP1 SUR LA REGULATION DE LA DIFFERENCIATION DES CELLULES SOUCHES MESENCHYMATEUSES HUMAINES
WO1998044142A1 (fr) * 1997-04-01 1998-10-08 The General Hospital Corporation Marqueur moleculaire pour cellules souches musculaires
WO1999020740A2 (fr) * 1997-10-23 1999-04-29 Geron Corporation Procedes et matieres utiles pour la croissance de cellules souches primordiales de primate

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HERRERA P. L. ET AL.: "Two transgenic approaches to define the cell lineages in endocrine pancreas delop'ment", MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 140, 1998, pages 45 - 50 *
JUN-HSIANG L. ET AL.: "A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice", PROC. NATL. ACAD-SCI. USA, vol. 92, 1995, pages 679 - 683 *

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ES2183665A1 (es) 2003-03-16
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ES2208024B1 (es) 2005-10-01
ES2183665B1 (es) 2004-08-01

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