ES2208024B1 - A METHOD FOR OBTAINING IN VITRO MOTHER CELLS OF INDIVIDUAL MAMMALS ADULTS. - Google Patents

Abstract

More specifically, the present invention relates to a method for obtaining in vitro stem cells from adult mammalian individuals, including the human species, which comprises gently disintegrating a tissue sample by using a buffered solution containing a reducing agent and a calcium chelating agent, culturing the disintegrated cells under conditions that keep said cells in an undifferentiated state, for a sufficient period of time for cell clones to appear, isolate and grow the obtained cells, and check their ability to give rise to tissues. With the cells obtained, cell and tissue banks can be generated for biomedical, biotechnological or research purposes.

Description

A method to obtain in vitro stem cells from adult mammalian individuals.

Field of the Invention

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 which are, obtaining and in vitro selection of differentiated cell types from of precursor cells. With the cells thus obtained, cell and tissue banks can be generated for biomedical, biotechnological or research purposes.

More specifically, the present invention relates to a method for obtaining in vitro stem cells from adult mammalian individuals, including the human species, which comprises gently disintegrating a tissue sample by using a buffered solution containing a reducing agent and a calcium chelating agent, culturing the disintegrated cells under conditions that keep said cells in an undifferentiated state, for a sufficient period of time for cell clones to appear, isolate and grow the obtained cells, and check their ability to give rise to tissues.

Background of the invention

Diabetes mellitus is a serious disease. metabolic with a huge socioeconomic impact, which affects between 10 and 20 million people in the European Union (about a 5% of the population) [Hauner et al., 1992; de Leiva et al., 1996 (see the section on Bibliography)] and with a estimated global incidence around 6% [Taylor et al., (1988); McGarry, (1992)]. All forms of diabetes are characterized by hyperglycemia derived from partial deficiency or total insulin, with the long-term appearance of alterations of the glomerulous area of the kidney and neurological complications [Williamson et al., (1993); Ruderman et al., (1992); Diabetes Control and Complications Trial Research Group, (1993)]. The diabetes mellitus also causes the development of pathology microvascular in the retina, currently being the first cause of loss of vision and blindness in adults. Diabetes mellitus insulin dependent (IDDM, also known as onset juvenile, or type 1) represents approximately 15% of all human diabetes Unlike what happens with diabetes Type 2, or non-insulin dependent (NIDDM) mellitus, type 1 involves the destruction of insulin producing beta cells of the islets of Langerhans in the pancreas. The destruction of beta cells in type 1 diabetes is considered the result of a autoimmune attack, in which the immune system itself patient recognizes and destroys his beta cells, although not the rest of cell types that complete the islet [Eisenbarth, (1986)]. TO Despite current treatment modalities, patients type 1 suffer episodes of blood glucose levels abnormally high (hyperglycemia) [Nathan, (1992)]. Those episodes uncontrolled and recurrent hyperglycemia are considered the cause of the development of secondary complications that lead to renal failure, blindness, neuropathy or diseases cardiovascular [Nathan, (1992)]. Glucose maintenance in blood under strict control is widely recognized as crucial in treating all forms of diabetes to delay the onset and reduce the severity of complications microvascular and some macrovascular complications [Diabetes Control and Complications Trial Research Group, (1991); Reichart et al., (1991); Nathan, (1993); Diabetes Control and Complications Trial Research Group, (1993)]. The biggest limitation of therapy Conventional insulin replacement, whether administered by injection or by mechanical devices, it is your inability to achieve a physiological control of insulin supply in response to changes in blood glucose. To try to approach this control, many patients are treated with an intensive regimen  of insulin administration. However, this intensive therapy of insulin can only be carried out in a subset of patients especially motivated Even this intensified form of insulin treatment only delays the onset of serious secondary complications of diabetes. Does not exist currently some form of insulin replacement therapy that prevent the development of diabetic complications.

Under those same circumstances, the provision of pancreatic beta cells can recover homeostasis from the glucose. An attractive alternative is tissue transplantation that produces insulin, which offers a much more approximation physiological for the precise recovery of homeostasis of the glucose, and therefore prevent the serious complications of diabetes. However, if islet transplantation becomes an extended treatment for diabetes, the supply of Human pancreas of organ donors will tend to be the factor limiting [Korbutt et al., (1997)]. In the transplants performed currently one or two full donated pancreas are required by transplanted individual Unfortunately, the number of pancreas donated is much lower than those required to treat patients Type 1 diabetics Therefore, of the millions of individuals diabetics who could benefit from such transplants, just a Very small number of them can be treated this way. In a attempt to overcome this supply problem, have been suggested two approaches: i) animal islet transplantation (xenotransplantation) between species phylogenetically more or less next [Lacy, (1993); Pipeleers et al., (1994)], or ii) engineering cell of insulin-producing tissue [Kawakami et al., (1992); Ferber et al., (1994); Balley and Docherty, (1994)], both by modification of beta cell lines [Efrat, (1998)], as per ectopic insulin production [Valera et al., 1994)].

Phase clinical trials are under way 1 islet transplant of porcine fetal Langerhans for diabetes mellitus treatment [Groth et al., (1994)], as well as porcine fetal central nervous system tissue for the treatment of Parkinson's disease [Isaacson et al., (1995); Deacon et al., (1997)]. The transplant has also been described Hepatic from baboons to humans [Starzl et al., (1993)] and the extracorporeal circulation to pig livers for treatment of fulminant liver failure [Charl et al., (1994)].

Even when the problems are solved of rejection and that the xenograft physiology can supply efficiently the transplant needs, still the serious risk of generating new ones would remain to be resolved human diseases This concern has been recently confirmed by the discovery that endogenous retroviruses of the pig can infect human cells [Patience et al., (1997)].

While the xenotransplant is limited by numerous physiological, immunological, ethical problems and Infectious, cellular engineering has so far oriented to prepare artificial beta cells from cells somatic

The beta cell lines could be modified to carry out the synthesis and secretion of insulin regulated by nutrients. For example, hamster beta cells (HIT cells) transformed with the SV40 virus [Santerre et al., (1981)], or BTC cells. The latter are the result of cell transformation using the SV40 T antigen oncogene functionally connected to the insulin gene promoter. 13 CT lines (tumor cell) derived from insulinomas that appeared in transgenic mice expressing the SV40 oncogene under the promoter of the insulin gene [Efrat et al., (1988); D'Ambra et al., (1990); Efrat et al., (1993); Knaack et al., (1994); S. Efrat, US Patent 5,723,333 (1998)]. These cell lines were established by transforming the cells with vectors, preferably retroviral vectors, containing two or more oncogenes under the control of one or more inducible promoters and / or genetic elements. A system that was improved by inserting a tetracycline-controlled genetic activator generated by the fusion of the tetracycline resistance operon encoded in the Tn10 genetic element of the Escherichia coli bacteria with an activation domain of the herpes virion protein 16 simplex virus [Gossen et al., PNAS 89: 5547 (1992)]. All these strategies use oncogeries inserted into the DNA to generate tumor cells, and even if the tetracycline-controlled genetic activator is introduced, a point mutation in this gene will cause a tumor. In addition, human beta cells are very difficult to transfect.

Modify autologous non-beta cells so that synthesize and secrete insulin would overcome the rejection immune In this sense, biosynthesis of insulin in various cell types, such as liver cells [Valera et al., (1994)], fibroblasts and muscle cells [Docherty, (1998); Gross et al., (1999)]. However, since these somatic cells do not have intracellular mechanisms to process and store insulin, these approaches produce a basal insulin production, sometimes transient, and not regulated, insufficient to maintain normoglycemia in animals diabetics, who need additional insulin supply. Newgard et al. have used a tumor cell line of origin endocrine, the AtT-20, to which the Insulin gene and other specific beta cell genes pancreatic such as Glut-2 or glucokinase [US patents US 5,427,940, US 5,744,327 and US 5,747,325]. Although iterative modification allows you to create a cell line that responds effectively to glucose, those cells Tumors can generate tumors and also express other hormones such as ACTH (adrenocorticotropic hormone), which in turn causes diabetes mellitus by itself.

Other possibilities to investigate include the generation of human beta cell lines from freshly born affected by nesidioblastosis and subsequent repair of defective genes that cause disease, or the discovery of the mechanisms and factors that control the maturation of Islets and regeneration from ductal cells [Macfarlane et al., (1997); Sharma et al., (1999)].

An alternative approach is the use of pluripotential stem cells to obtain in vitro cells and tissues for transplantation. The success in the culture of human pluripotential embryonic cells [Gearhart, (1998)] opened great expectations for the treatment of neurodegenerative diseases, diabetes, spinal cord injury, hematopoietic repopulation, or repair and regeneration of damaged tissue. Likewise, 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. However, the conditions for inducing targeted 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. New strategies have to be developed to obtain the large amounts of pure cell populations necessary for transplantation. So far, attempts to prepare cells from pluripotential embryonic cells in order to obtain pure populations of cells of a single phenotype with therapeutic applicability have failed. Methods that employ an external marker of a differentiated cell type are not capable of producing a pure and abundant population of cells. Other more elegant methods are based on a strategy that allows to follow and characterize cell lineages and branching points in the differentiation process. Briefly, a marker gene functionally connected to regions of DNA regulating development-dependent expression and cell lineage is introduced into pluripotential cells before cell differentiation.

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 certain cells towards the neuronal lineage that continues until terminal differentiation in culture. Based on this strategy, 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 immunocytochemistry. While this latter method can be carried out in vitro , it has the disadvantage that it only allows isolating abundant populations in the culture of early differentiation cell lineages, which excludes cell lineages that appear in late stages of development. In addition, since the method is restricted to those proteins that can be followed by FACS or by immunocytochemistry, it does not result in the isolation of pure populations. This method, which has been poorly demonstrated experimentally, represents an advance over previous work, although:

- can only be applied to cells embryonic pluripotentials;

- the separation method, FACS or immunocytochemistry only allows separating populations present in large numbers, and not those that represent a fraction marginal (1 in a million or less);

- the method preferably uses proteins membrane expressed markers; Y

- the method is restricted to the Otx, Dlx genes, Nlx, Emx, Wnt, En, Hox, and ACHR13, which are genes that encode for large groups of cell lineages and are not specific to a  specific cell type.

Accordingly, there is a need to develop a method for differentiating and isolating in vitro both precursor cells determined to a specific cell line and terminal differentiated cells, with a specificity sufficient to select in vitro 1 cell from at least 10 7. In addition, it would be desirable that such a procedure:

a) allow to obtain specific cell types starting not only from pluripotential embryonic cells, but also from individual cells, which would open the possibility of new therapeutic and biomedical applications, by example, obtaining specific differentiated cells from of precursor cells of the patient himself; I

b) allow to obtain specific cell types differentiated enough not to have the potential of stem cell proliferation, and therefore the ability hypothetical of getting to produce tumors in the individual transplanted; I

c) provided with biological safety systems that prevent contamination of cells with other unwanted cell types, advantageously, it should allow additional biosecurity systems to be inserted into the cell, for example, the thymidine kinase gene that confers ganciclovir sensitivity , in order to remove differentiated cells pharmacologically, both in vitro or in vivo ; I

d) allow to obtain specific cell types  differentiated that have already demonstrated their therapeutic potential.

In addition, the possibility of a differentiated cell type with utility Therapeutics can be obtained from another adult cell. This is a new postulate that contrasts with the accepted process of embryonic development. Cellular differentiation is the process by which embryonic cells become different from each other, acquiring different identities and specialized functions [Wolpert, (1998)]. Initially the cells are genomically equivalent, and are programmed differentially during the individual development In mammals there are more than 200 types distinct cell phones clearly recognizable. The cells differentiated perform specialized functions and have reached a stable terminal state. The central process in the Cellular differentiation is the change in gene expression. It entails the production of specific cell-type proteins that characterize a totally differentiated cell, for example the hemoglobin in red blood cells, insulin in beta cells pancreatic, etc. In early stages the differences between cells are not easily detectable, they are probably changes in one or two genes However, once determined towards a destination specifically, the cell passes from that determined state to give rise to All his progeny. Frequently, the determined cells experience several rounds of undisclosed cell divisions in its external appearance, its identity and / or destiny. Daughter cells inherit the parental cell differentiation program until that program (terminal differentiation) is executed after a considerable period of time. A central feature of development and cell differentiation states that once the differentiation program is installed and the terminal differentiation, the state of differentiation of that cell is maintained throughout his life, and that although in the individual adult there are some stem cells that maintain the ability to form new tissues, this is restricted to those tissues that require continuous regeneration, such as the skin, intestine, or the epithelium of the cornea. It is also widely accepted that others tissues can initiate regeneration under certain conditions, Like the liver There are some exceptions to this rule. The classical transdifferentiation experiments [Okada, (1980); Schmidt and Alder, (1984); Okada, (1991); Anderson, (1989); Eguchi and Kodama, (1993)] and the most recent cloning in sheep and mice to starting from adult cell nuclei suggest that the cells of adults can be tested as donors to create new types Cellular with therapeutic utility.

Transdifferentiation 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 unexpected and ectopic tissues. Metaplasia can appear as a result of selective growth of very minor cells present in a certain organ. In other cases, the new differentiation may occur as a result of the re-differentiation of pre-existing differentiated cells.

Transdetermination (Hadorn, 1978) 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.

Dedifferentiation 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].

Recent experiments in which it has been managed to clone a complete mammal from the nucleus of a adult cell suggests that differentiated cells retain the ability to reactivate repressed genes and return to the state pluripotential. This implies that cell differentiation does not implies irreversible modification of the genetic material required to develop a mammal in term. This has been Shown in sheep and mouse. The possibility of that a small proportion of relatively stem cells undifferentiated are in the breast cell to allow their regeneration during pregnancy. However, in terms of their Biotechnological applications, it is not relevant that the nucleus proceed of a stem cell of an adult individual or of a cell quiescent in the Go phase of the cell cycle of the same individual. The which is essential is to know if a cell of an adult mammal can generate other adult tissue like pancreatic beta cells, photoreceptors or hepatocytes, or even any of them, to to have new therapeutic tools with which be able to treat virtually any somatic disease.

Therefore, there is a need to develop a new procedure to generate differentiated adult cells to  from other adult cells, which can be generated patient's own cells with therapeutic utility avoiding explicitly and therefore eliminating the hypothetical need for create pluripotential embryonic cells using techniques of cloning On the other hand, these cells could be used to generate cellular and tissue banks for emergency situations, as for example, before a fulminant liver failure or while is on the waiting list for a transplant.

An object of this invention constitutes a DNA segment, useful for selecting cell types defined differentials, comprising (i) the regions that they contribute to form and stabilize the complex of factors of transcription of a specific gene of a cell type that is specifically expressed in a differentiated cell type, and (ii) a functionally connected selection gene, so that it express when the DNA segment is recognized and transcribed by the complex of transcription factors 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 differentiated cell type. A method to mature in vitro until terminal differentiation the differentiated cells obtained also constitute an object of this invention.

Precursor cells transfected with said DNA segment as well as the differentiated cells obtained at from said transfected precursor cells constitute additional objects of the present invention.

Other additional objects of this invention are methods to obtain stem cells from adult mammalian individuals, as well as certain stem cells, which comprise the use of said segment of DNA.

More specifically, the present invention has as an object:

- a method for obtaining in vitro stem cells from adult mammalian individuals, including the human species;

- an adult mammalian stem cell obtainable by said method for obtaining in vitro stem cells from adult mammalian individuals;

- a precursor cell, obtainable by said method for obtaining in vitro stem cells from adult mammalian individuals, transfected with said segment of DNA useful for selecting defined distinct cell types;

- a method for maturing in vitro to terminal differentiation to a cell obtainable by said method for obtaining in vitro stem cells from adult mammalian individuals; Y

- the use of these cells for the study of the processes that lead to differentiation and cell dedifferentiation.

Brief description of the figures

Figure 1 is a schematic representation of the DNA segment used in the embodiment of Example 1. The arrows indicate the approximate cutting sites of the enzymes of corresponding restriction.

Figure 2 is a graph representing the blood glucose level at different times in animals transplanted and in controls [see Example 1].

Figure 3 is a graph showing the evolution of body weight over time in animals transplanted and in controls [see Example 1].

Figure 4 is a graph that represents the glucose tolerance test results at different times in transplanted animals and controls [see Example 1].

Figure 5 are photomicrographs of a section histological spleen of a representative transplanted animal, analyzed by immunohistochemical techniques that reveal the presence of insulin as ocher color staining [see Example one]. Figure 5A shows a diabetic pancreas section without islets and Figure 5B shows a transplanted spleen section.

Figure 6 are cut photomicrographs histological tissue derived from transplanted lines in immunosuppressed mice [see Example 3], specifically, of mesodermal tissue (Figure 6A), of tissues endodermal and mesodermal (Figure 6B), and of endodermal tissues, ectodermal and neuroectodermal (Figure 6C).

Detailed description of the invention

The techniques described below, such as the generation and use of expression vectors, cell cultures, transfection, selection and growth of clones  Cellular, obtaining diabetic animals by injection of Streptozotocin, or cell and tissue transplantation are known and routinely carried out by staff with a preparation ordinary in this field of research. Unless specified otherwise, all technical and scientific terminology has the same meaning as commonly understood by a person with an ordinary preparation in the. research field to which This invention belongs.

1. The DNA segment

Transcriptional control is crucial for the cell differentiation since it determines which genes are transcribed and therefore what proteins the cell will produce. The gene expression is regulated by the coordinated action of regulatory proteins (transcription factors) that bind to key regions of DNA for control of gene expression. The interactions of transcription factors can be very complex and the number, type and combinations of proteins regulators that form the transcription complex is known only very partially and in a few cases. Therefore, the possibility of directing cell differentiation using this Little knowledge is excessively limited.

In the present invention a much simpler alternative. Although a single protein Regulatory may participate in more than one transcription complex, 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 segment of DNA that is recognized by the transcription complex, may be express any gene (for example a selection gene) inserted functionally to that segment of DNA only when the combination of factors that form the transcription complex of the segment of DNA found in the cell. The key point is to build a DNA segment that is recognized by the transcription complex of a specific gene of a particular cell type that allows select said cell type. As an example, the segment of DNA that allows successful selection of pancreatic beta cells at from mouse pluripotential cells, from human teratocarcinoma, and murine adult intestinal cells and human should be recognized by the transcription complex of insulin gene Preferably, the specific gene used must be of the species to which the cells belong precursors

It is understood by "transcription factor" any protein required to initiate or regulate the transcription. It includes both specific regulatory proteins of group of genes as common proteins of the general machinery of transcription. The "transcription factor complex" is the  group of factors that recognizes the DNA regulatory regions of A gene to start transcription. "Regulatory regions of DNA "include not only binding sequences for transcription factors, identified or not, but also other regions that have a relevant role in DNA binding of complex of transcription factors.

It is understood by "segment of DNA", in In general, any exogenous DNA that contains a selection gene functionally connected to the DNA regulatory regions of a specific gene This concept is especially relevant in the Development of the present invention. This fragment of regions of DNA called DNA segment is defined exclusively for the final result of the selection of the specific cell type With a defined phenotype.

The invention provides a useful DNA segment. to select differentiated cell types, sometimes called the DNA segment of the invention, which comprises (i) the regions that contribute to form and stabilize the complex of transcription factors of a specific gene of a cell type which is specifically expressed in a differentiated cell type, and (ii) a functionally connected selection gene, so that express when the DNA segment is recognized and transcribed by the complex of transcription factors of the specific gene.

The specific gene is a gene that expresses itself. preferably in a single differentiated cell type, although You can also use specific genes that are expressed in more than a differentiated cell type even if they do it preferably in a concrete differentiated cell type. Therefore, in general, any specific gene of a cell type that is expressed specifically in a differentiated cell type it can be used in the DNA segment of the invention. In one embodiment particular of this invention, said specific gene of one type cell that is specifically expressed in a cell type differentiated is a selected mammalian gene, including the man. Thus, the insulin gene would be used to obtain the pancreatic beta cells, the rhodopsin gene for photoreceptors of the retina, the ornithine gene transcarbamylase for the liver or albumin gene, these being understood as non-limiting examples of genes specific to differentiated cell types.

The selection gene is a gene whose expression provides a phenotypic feature that allows you to select cells transfected with said selection gene. Can, therefore,  use any selection gene that meets the condition previously established, in the DNA segment of the invention. In a particular embodiment of this invention, said gene of selection is selected from a resistance gene, for example, the gene for resistance to geneticin, the gene for resistance to dihydrofolate, the puromycin resistance gene, the gene of gamma interferon resistance, and the resistance gene to methotrexate (multidrug resistance gene), a temperature gene and your combinations

How the specific gene can be expressed in different stages of development, but they will only be selected those cells determined to be a specific cell type, a strategy to increase the specificity of the procedure to the selection of said cell type implies the use of a gene secondary that is also expressed in the specific cell type. After insertion of another positive selection gene, different from that is functionally connected to the specific gene, in a region of the secondary gene that allows expression of the second gene of selection when the DNA regions of the secondary gene are recognized for its complex of transcription factors, it introduce the specific and secondary genes, and their respective selection genes, in the precursor cell to differentiate. In this In this case, cell type selection could be done through a double positive selection. The secondary gene can be expressed in other cell types, but the combination of gene expression specific and secondary gene must be exclusive to the phenotype of cell type that is intended to be obtained.

Therefore, the invention also provides a DNA segment of the invention, further comprising a gene secondary that is expressed in the cell type in which it is expressed the specific gene, functionally connected to a second gene of selection, different from the selection gene connected to the gene specific. Preferably, said DNA segment comprises, in addition, the region that contributes to form and stabilize the complex of transcription factors of said secondary gene of the type cell to select.

Any secondary gene of a cell type in the that specifically expresses a specific gene can be used in the DNA segment of the invention as long as the  combination of specific gene expression and gene secondary is exclusive to the phenotype of the cell type that pretend to get. In a particular embodiment, said gene secondary is selected from the group formed by the gene of the glucokinase or the glucose transporter gene Glut-2 for pancreatic beta cells and the gene of the phosphoenolpyruvate carboxykinase or the albumin gene for the liver.

The invention also contemplates the development of a DNA segment of the invention further comprising 2 or more non-specific genes of the cell type to select but whose combined expression is specific to the cell type to select, Operationally connected with its regulatory regions corresponding, and each of said regulatory regions being functionally connected with at least one selection gene.

The DNA segment of the invention may also advantageously contain a biological safety system that prevents contamination of the 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 remove manipulated cells pharmacologically, in vitro or in vivo .

General techniques for obtaining the DNA segment of the invention, microbiological techniques, cloning and DNA manipulation techniques, electrophoretic techniques, etc. they are those described by Ausubel et al. in the Current Protocols in Molecular Biology laboratory manual, Ed. John Wiley & Sons Inc., New York (1995); and by Sambrook et al. in the book Molecular Cloning: A Laboratory Manual , Ed. CSHL Press (1989).

2. Procedure for obtaining a cell type differentiated

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) cultivate the transfected precursor cells under appropriate conditions for them to become the type differentiated cell;

c) select the differentiated cell type that expresses the specific gene and, consequently, the selection gene; Y

d) cultivate and grow the differentiated cell type until reaching the required amount.

Precursor cells, before being transfected with the DNA segment of the invention, they are cultured under conditions that guarantee that they remain in state undifferentiated, for example, by adding to the medium of cultivation [such as Dulbecco-modified DMEM)] 15 to 20% of fetal bovine serum and factors that inhibit differentiation. spontaneous cells, such as the inhibitory factor of leukemia (1,000 U / ml, ESGRO, Life Technologies, Inc.). The undifferentiated precursor cells, in a number of approximately 10 5 -10 7 cells per transfection session, they are transfected with a segment of DNA from the invention.

Suitable conditions for culturing 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 groups of aggregate cells (embryonic bodies) so that certain cells appear towards multiple cell lineages; for this the precursor cells are cultured, 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)].

Then the bodies are placed embryonic in culture plates to which they can adhere and select the differentiated cell type that expresses the gene specific, and, consequently, the selection gene, by means of modification of culture conditions (for example, by the addition of a cytotoxic agent for 1-2 weeks) that will eliminate any cell that does not have the gene activated specific, thus only the cell type of interest will survive and phenotypically homogeneous cultures will be available in terms of The expression of the specific gene.

Next, the differentiated cell type selected is grown and grown, for example, under conditions of adherent culture (monolayer plate culture) until reaching amount needed (usually from 10 4 to 10 10 cells) to  obtain effective therapeutic response, to perform trials of research, to produce biotechnological derivatives, or to Any other type of application.

Once the necessary number of cells is obtained, terminal differentiation can be initiated, in the event that the cell type sought possess that characteristic (for example, in the case of obtaining pancreatic beta cells, neurons, cardiomyocytes, corneal epithelial cells, cells of the blood, chromaffin cells of the adrenal medulla or photoreceptors of the retina). The conditions for the differentiation to begin terminal will vary depending on the cell type, for example, adding differentiating factors to the culture medium; so for get neurons retinoic acid and adenosine monophosphate will be added cyclic (cyclic AMP); dimethylsulfoxide (DMSO) will be added to obtain cardiomyocytes; glucocorticoids will be added to obtain chromaffin cells; or nicotinamide will be added to obtain cells pancreatic beta

Also, 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, for example, the gastrointestinal epithelium, skin cells, epithelial cells of the ocular limbus. , mammary gland cells, liver cells, mucosal epithelial cells, hematopoietic cells, ependymocytes, or pancreatic ductal cells. For this, 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 culturing for several days (1-3 weeks) under conditions that keep the disintegrated cells in an undifferentiated state [as previously mentioned, for example, by adding bovine fetal serum culture medium and factors that inhibit spontaneous differentiation of cells]. After this period of culture cell clones appear, and after isolation and growth their ability to give rise to tissues is checked. For this, 10 6 -10 7 cells are inoculated subcutaneously in immunosuppressed mice. After 2-3 weeks the different tissues that these cells have formed are analyzed, and the degree of pluripotentiality that the generated precursor cells possess is verified. The greater the number of cell types and differentiated tissues obtained, the greater the degree of pluripotentiality of the precursor cells. Cells that only give one type of tissue can preferably be used to obtain the differentiated cell types that make up that tissue, while cells that give rise to multiple tissues can be used as precursor cells to obtain a greater number of cell types

Through the process of the invention you can Obtain any of the more than 200 cell types differentiated that make up the human body (as an example, see the cell types described above) as well as cells precursors of adult individuals who are determined to give place to a specific lineage. For example, in the case of blood cells, precursor cells of the red series, monocyte precursors, neutrophil precursors, or precursors of lymphocytes; in the case of glial cells, precursors of astrocytes, oligodendrocytes, schwann cells, cells ependymal or microglia; in the case of neuronal cells, precursors of the different specialized types of neurons present in the seven anatomical regions of the nervous system central, or in the case of the cells that make up the islets pancreatic, precursor cells of endocrine types specialized (glucagon or beta or alpha producing cells insulin producers, delta or somatostatin producers, or PP cells).

When differentiated cells are obtained they are from an individual, the process of the invention allows to create  differentiated cells with the immunological characteristics of own individual. Such personal cells would be suitable for his eventual transplant to the individual and his employment would avoid Immune rejection problem in case of autotransplantation.

The process of the invention opens the possibility of obtaining biological material from the individual to the effective cell therapy of many diseases, practically of any somatic disease, for example, diabetes mellitus, Parkinson's disease, retinitis pigmentosa, etc.

In addition, the process of the invention is a continuous source of cells of a certain selected type, what which allows new therapeutic approaches for transplants, for example, liver or pancreatic transplantation, 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 further illustrated. below with some concrete examples of obtaining beta cells pancreatic that can be used in multiple applications, for example, in the treatment of diabetes mellitus (both type 1 as type 2), in the detection of antigens associated with diabetes, and even in large-scale insulin production properly packaged

The process 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. On the other hand, 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.

Since it is a universal procedure, it includes the generation of any cell type and its applications, by example, biomedical or research.

3. 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 They really have them. Initially, it was called "cell mother "to a population of precursor cells that had a large self-maintenance capacity [Lajtha, (1970)]. One more definition recent distinguishes between active, potential stem cells, lineage-specific and determined [Potten and Loeffler, (1990); Potten, (1996)]. "Active stem cells" are cells undifferentiated precursors capable of proliferating, self-sustaining, produce a large number of derived cell lineages, regenerate tissues after an injury, and have flexibility in the use of those capacities "Potential stem cells" are the version latent or quiescent active stem cells. The cells certain mother "are transient cells capable of division and that they can share with the cells terminally differentiated the ability to perform specific functions of tissue. 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 type mobile. In this sense it is different from the "stem cell lineage specific ", which is a specific precursor cell 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  differentiate only after having multiplied. These adult stem cells are responsible and necessary for the tissue growth during development, regeneration of certain organs such as the liver, or the continuous production of blood or skin cells The results obtained by inventors indicate that, under certain conditions, the cells Adult precursors can also be pluripotential. "Pluripotential stem cell", a term that has been restricted so far 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 a embryo. The "immortal stem cell" is defined as a cell population that permanently retains its characteristics embryonic and that can be multiplied in culture unlimitedly, or differentiate multiple lineages and cell types under certain  terms. According to the results of the inventors, the origin of these immortal stem cells can be a stem cell as an adult

To carry out the procedure of the invention any precursor cell can be used, by for example, stem cells, pluripotential embryonic, embryonic carcinoma cells, or cells of human individuals adults who, under defined cultivation conditions, behave as stem cells (adult stem cells), for example, cells of the gastrointestinal epithelium, skin cells, cells epithelial of the ocular limbus, mammary gland cells, liver cells, mucosal epithelium cells, cells hematopoietic, ependymocytes, or pancreatic ductal cells.

4. Verification of the pluripotentiality of a cell adult mother

As indicated above, there is a simple experiment to check the possibility of preparing new cells and tissues using cells obtained from adult individuals as donors. Briefly:

a) one or more cell lines are generated to from a sample of adult tissue (see examples described above)

b) a certain number of these are transplanted cells (one to ten million) to immunosuppressed mice for avoid immune rejection; Y

c) analyzed after several days of transplant the types of tissues formed.

The results obtained by the inventors [see Example 3] show that cell lines generated from adult mammals (mouse and human) have the potential of giving rise to cell types and tissues (neuronal tissue, epithelium mucosecretor, striated muscle, cartilage, bone, etc.) derivatives of the three embryonic layers (ectoderm, mesoderm and endoderm).

5. Transfection and cell selection

The term "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 [Ausubel et al., Current Protocols in Molecular Biology , Ed. John Wiley & Sons Inc., New York (1995); and Sambrook et al., Molecular Cloning: A Laboratory Manual , Ed. CSHL Press (1989)].

In a particular embodiment of this invention, cell type selection from stem cells embryonic or adult allows to select stem cells determined to be pancreatic beta cells.

Embryonic development follows a model exquisitely organized in which a large number of factors, many still unknown, and the cell biography itself determines the differentiation path that a certain cell will follow. Low certain growing conditions [Roberston, (1987)], the possibility of a stem cell becoming determined after a Asymmetric division is random. The selection of a certain cell type allows to isolate, under controlled conditions, those cells that meet two requirements: ability to divide and the specific gene expression. The proportion of these cells present in the culture prior to selection is extremely low, so the process of the invention must be very powerful and specific enough not to give false positives. When using adult stem cells to generate a cell line used to produce pancreatic beta cells the process is similar to that described previously.

In a particular embodiment of the procedure of the invention, the selection of the differentiated cell type that Express the specific gene is performed in culture.

Alternatively, the selection of said type differentiated cell expressing the specific gene can be performed  through a positive double selection procedure using a segment of DNA of the invention comprising a secondary gene, functionally connected to another selection gene other than connected to the specific gene, in addition to the specific gene.

6. Maturation of the pancreatic beta cell

When the specific gene corresponds to a cell  terminal differentiated, the selected cell must be a determined stem cell that maintains its ability to divide until other factors cause their terminal differentiation. In In the case of the pancreatic beta cell, there are numerous factors extracellular that inhibit their proliferation [Hügl et al., (1998)]. The most effective are the inhibitors of phosphoproteinophosphatases, such as orthovanadate, or inhibitors of intracellular signals mediated by protein kinases that phosphorylate tyrosine residues, such as genistein, wortmanin, and the compound 2- (4-morpholinyl) -8-phenyl-4H-1 -benzopiran-4-one identified as LY294002 [Vlahos et al., (1994)].

The process that leads to cell maturation Beta is not well known and is an active field of research. This process involves both cell proliferation and differentiation terminal. Since pancreatic beta cells do not divide, This process requires opposite behaviors in practice. Some factors such as betacellulin, the growth factor of alpha tumors, nerve growth factor, or activin A activate proliferation, while nicotinamide, butyrate sodium, vitamin D 3 or genistein inhibitors and Wortmanin stop proliferation. For example, nicotinamide causes growth and maturation of pancreatic beta cells [Otonkoski et al., (1993); Anderson et al., (1994)], and of the 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 beta cells pancreatic [Lee et al., (1994)].

Example 1 that accompanies this description demonstrates that certain stem cells obtained after selection of cells that express the specific gene were already beginning to manifest incipient phenotypic characteristics of cells pancreatic beta (insulin content, insulin release at  means, medium). However, only after 1-3 weeks in a nicotinamide differentiation culture medium 5-25 mM followed by 1-3 weeks in other culture medium of terminal differentiation with glucose 3-5 mM and nicotinamide 5-25 mM se generated a pure cell population that had a content of insulin just like the mature pancreatic beta cell, which responded  efficiently and physiologically to secretagogues just like the mature pancreatic beta cell, and that did not divide, nor They do mature beta cells.

7. Other applications of the invention

The present invention is universal in nature, so it 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, alterations musculoskeletal, hepatopathies, immunodeficiency syndrome. acquired, non-regenerative anemias, etc.), obtaining products derivatives of specific cell types for the development of products useful in biomedical, biotechnological or investigation; for example, obtaining drugs of origin biological for blocking tumor dedifferentiation, or anti-idiotype antibodies for blocking immune reactions undesirable, obtaining cellular antigens for the detection of useful antibodies for the diagnosis of diseases; obtaining of biological reagents for research aimed at development of new diagnostic procedures, prognoses or disease treatment; use of the cells obtained by the  procedure of the invention for the study of the processes that lead to differentiation and cell dedifferentiation, particularly in the study of the tumor process.

Therefore, transfected precursor cells with a segment of DNA of the invention, as well as the cells differentiated obtained from said precursor cells they constitute additional objects of the present invention.

The invention also provides a method for culturing in vitro differentiated cells in liquid medium, for example, pancreatic beta cells obtainable by the process of the invention, which comprises 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 obtain a product of interest comprising the expression of differentiated cells obtainable by the procedure of the invention. In a particular embodiment, said product of interest is selected among drugs of biological origin for blocking of tumor dedifferentiation, anti-idiotype antibodies for blocking of undesirable immune reactions, cell antigens for the detection of antibodies useful for the diagnosis of diseases, and biological reagents for research aimed at the development of new diagnostic procedures, prognosis or treatment of diseases. In one embodiment concrete of this invention, said product of interest is the insulin, and is obtained by peripheral beta cells pancreatic obtainable by the method of the invention in a Buffered liquid medium containing secretagogues.

All these applications are possible since the process described in the invention allows to obtain large amounts of cells (10 10 cells or even more) of specific cell types in vitro that are normally found in much smaller proportion in adult individuals. Likewise, cadaveric donation, or the presence of potentially transmissible infectious agents in the production of biological derivatives, are avoided.

The invention provides a method for obtaining in vitro 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 certain precursor cells of adult mammalian individuals, including the human species, which comprises transfecting cells of said adult mammalian individuals with the DNA segment of the invention, culturing the transfected cells under conditions suitable for convert into determined 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 DNA segment of the invention, of cell types differentiated obtained by the process of the invention, optionally matured until terminal differentiation, of the precursor cells transfected with the DNA segment of the invention, in all kinds 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. Is predictable that staff with ordinary preparation in this field 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, by therefore, not limiting the scope of it.

Example 1 Obtaining and therapeutic evaluation of pancreatic beta cells differentiated from stem cells (or precursor cells) mouse embryonic 1.a) Murine embryonic stem cell culture (CMEM)

The culture of the CMEM was carried out following standardized protocols [Robertson, (1987)].

1.b) DNA segment transfection

The segment of DNA whose characteristics are shown in Figure 1 CMEM was transfected by electroporation or lipofection, obtaining similar results in both cases, and culture of the transfected cells during 24-48 hours without any selection. The segment of DNA includes the regions that contribute to form and stabilize the complex of human insulin gene transcription factors (specific gene), a gene that confers antibiotic resistance geneticin (selection gene) functionally connected to the gene of the human insulin, a constitutive selection gene (gene of hygromycin resistance) which allows the selection of cells that have incorporated the DNA segment and the gene from the thymidine kinase (TK) that confers sensitivity to ganciclovir (ganc) (biosecurity system). The DNA segment was obtained through general molecular biology techniques, as has been indicated above.

1.c) Selection of transfected cells

The selection of transfected cells is 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 clones isolated gave similar results, and all of them differentiated  mature pancreatic beta cells.

1.d) Induction of in vitro differentiation of hygromycin antibiotic resistant clones

Cellular aggregates were generated by culture in suspension, following standardized protocols [(Robertson, (1987)]. After 5-7 days, cell aggregates were distributed in gelatinized tissue culture plates and they were kept in culture for another 3-7 days.

1.e) Selection of cells expressing insulin

The selection of insulin-expressing cells from differentiated cell clones in vitro was performed by adding to the culture medium of the antibiotic geneticin (0.2 mg / ml) for 10-14 days, followed by the expansion of resistant cells appeared.

1.f) Induction of in vitro terminal differentiation of selected cells expressing insulin

Cellular aggregates were generated (containing 10 2 to 10 3 cells per aggregate) following the protocol indicated in 1.d). After 2 days in culture, it was added to the medium of 20 mM nicotinamide culture and the cultivation of the cell aggregates for 2 weeks. Then, the usual culture medium (minimum essential medium modified by Dulbecco, or DMEM) to the terminal differentiation medium consisting on a medium basis CMRL-1099 (Life Technologies) supplemented with 20 mM nicotinamide, and the cells were maintained in culture for another 7-14 days. Table 1 shows insulin content and secretion based on evolution of the differentiation of several cell clones obtained by means of the Procedure of Selection of Cellular Types object of The present invention.

TABLE 1 Insulin content and secretion depending on the differentiation of several independent cell lines obtained by the Type Selection Procedure Cell phones

Grade of Line of Content from Secretion Secretion differentiation cells insulin basal from stimulated (ng / \ mug prot) insulin (pg / \ mug prot) (pg / \ mug prot) Cell isolated H1 0.4 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 without H2 0.5 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 differentiation H3 0.5 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 terminal [1.e)] R1 0.3 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 R2 0.3 ± 0.1 0.4 ± 0.1 0.8 ± 0.1 R3 0.3 ± 0.1 0.4 ± 0.1 0.7 ± 0.1 Cell added H1 16.1 ± 1.4 81.0 ± 5.8 511.8 ± 31.9 in H2 17.3 2.7 72.7 ± 3.5 481.2 ± 20.1 differentiation H3 16.2 ± 2.1 75.2 ± 3.9 519.3 ± 42.6 terminal [1.f)] R1 15.5 ± 1.1 71.2 ± 2.2 441.9 ± 38.1 R2 18.1 ± 2.5 86.9 ± 4.4 602.6 ± 44.0 R3 16.9 ± 1.9 79.5 ± 3.1 535.9 ± 59.4 Control Cell 20.1 ± 1.0 95.1 ± 2.3 655.3 ± 18.7 positive beta from pancreas from adult

1.g) Therapeutic evaluation of beta cells pancreatic, obtained from CMEM through the Procedure of Cellular Type Selection

For therapeutic evaluation of cells pancreatic beta obtained from CMEM through the Cell Type Selection Procedure the test was performed described below. 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 during at At least three days, and they had large amounts of glucose and ketone bodies in urine determined by test strip urine (+++) (Amestix, Ames). Differentiated cells (from section 1.f) 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 Subsequently, glucose was determined in blood and weight of transplanted animals with a frequency daily for the first two weeks, and then twice for week. The animals experienced a progressive reduction in hyperglycemia until normalization of the values of blood glucose in the first week after transplantation, and gained weight until they reached normal body weight [see Figures 2 and 3].

In order to confirm the therapeutic value of transplanted cells in the recovery of homeostasis of glucose, glucose tolerance tests were performed on the  transplanted animals at two weeks and one month after performed the transplant. The animals that are given administered the cells presented a tolerance test to the normal glucose as well as healthy animals that were used as a control [see Figure 4].

In order to verify the presence and activity of the transplanted cells in animals, some of the mice were sacrificed, and analyzed by immunohistochemistry both the spleen, where their cells were transplanted, such as the pancreas, to verify that they did not exist normal endogenous islets, and that, therefore, the disappearance of diabetic process was due to the administration of the cells beta obtained by the Cell Type Selection Procedure  provided by this invention [see Figures 5A and 5B].

Example 2 Obtaining and therapeutic evaluation of pancreatic beta cells differentiated from human teratocarcinoma cells 2.a) Establishment and culture of stem cells (or precursors) of human teratocarcinoma obtained from a testicular tumor (CTCE)

The establishment and culture of stem cells from human teratocarcinoma obtained from a testicular tumor (CTCE) was carried out following standardized protocols [Robertson, 1987].

2b) -2f)

The procedure was followed equally described in sections 1b) -1f) of Example 1. The Table 2 shows the content and secretion of insulin before and after differentiation of several cell clones obtained by means of the Procedure of Selection of Cellular Types object of The present invention.

TABLE 2 Insulin content and secretion depending on the differentiation of several independent cell lines obtained by the Type Selection Procedure Cell phones

Grade of Line of Content from Secretion Secretion differentiation cells insulin basal from stimulated (ng / \ mug prot) insulin (pg / \ mug prot) (pg / \ mug prot) Cell isolated TCX1 0.3 ± 0.1 0.3 ± 0.1 0.6 ± 0.1 without TCX2 0.4 ± 0.1 0.4 ± 0.1 0.7 ± 0.1 differentiation TCX3 0.5 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 terminal Cell added TCX1 12.5 ± 1.1 58.4 ± 3.6 380.9 ± 28.8 in TCX2 13.7 ± 1.4 61.1 ± 2.3 401.5 ± 40.1 differentiation TCX3 10.2 ± 0.9 55.9 ± 4.2 338.1 ± 36.7 terminal

As a positive control beta cells were used Adult mouse pancreatic The results obtained were not significantly different from those indicated in the Table one.

2.g) Therapeutic evaluation of beta cells pancreatic obtained from human teratocarcinoma cells through the Cellular Type Selection Procedure

The therapeutic evaluation phase is currently under way in vivo in immunosuppressed diabetic mice to which the beta cells obtained by the Cell Type Selection Procedure provided by this invention will be administered.

Example 3 Obtaining and assessing stem cells (or precursors) of adult from a sample of intestinal epithelial cells human 3.a) Establishment and culture of adult stem cells human (CMAH) obtained from a sample of cells human intestinal epithelials

The establishment and culture of stem cells from human adult (CMAH) obtained from a sample of cells Human intestinal epithelial was carried out following the. protocol described below:

1) incubation for one hour at 4 ° C of the sample in physiological saline to which it was added 10 mM dithiothreitol;

2) removal of the incubation medium and substitution by means of disintegration, consisting of a standard buffered saline (PBS) to which they are added 10 mM dithiothreitol and 2 mM ethylenediaminetetraacetate;

3) incubation for 1 hour at 4 ° C in the middle of disintegration in continuous agitation;

4) recovery of isolated cells in suspension by centrifugation at 1500 rpm for 5 minutes, and elimination of the disintegration medium;

5) resuspension in isolation, composed of DMEM standard medium supplemented with 15% serum heat-inactivated bovine fetal, a standard mixture of 100 mM non-essential amino acids (Life Technologies), 0.1 mM 2-mercaptoethanol, 10 micrograms / ml of Streptomycin, 10 Units / ml of penicillin and 10 micrograms / ml of insulin;

6) distribution of cells in petri dishes  gelatinized, which are kept in culture until the appearance of cell clones (1 to 3 weeks); Y

7) clone isolation and expansion cell phones obtained. Each of the expanded clones results in An independent cell line.

3.b) Evaluation of the potential of the lines of CMAH

In order to verify the potential of the CMAH lines, were transplanted to immunosuppressed mice at the level  106 subcutaneous cells per mouse and per CMAH line analyzed, in single session After 2-3 weeks the transplant by obtaining biopsy and examination pathological

Examination of the samples obtained from the lines of CMAH indicated that two of the six lines analyzed in this first  study resulted in. tissues derived from the three layers embryonic, ectoderm, mesoderm and endoderm [see Figures 6A, 6B and 6C]. These results clearly indicate that the two CMAH lines obtained from the intestinal epithelium sample adult human really behave like stem cells active pluripotentials.

Example 4 Obtaining and therapeutic evaluation of pancreatic beta cells differentiated from adult stem cells (or precursors) derived from a sample of intestinal epithelial cells murine 4.a) Establishment and culture of adult stem cells murine (CMAM) obtained from a sample of cells mouse intestinal epithelials

The establishment and culture of stem cells from murine adult (CMAM) obtained from a sample of cells Mouse intestinal epithelial was performed following the protocol detailed in sections 3.a) and 3.b) of Example 3.

4b) -4f)

The procedure is followed in the same way described in sections 1.b) -1.f) of Example 1, 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 means of the Procedure of Selection of Cellular Types object of The present invention.

TABLE 3 Insulin content and secretion depending on the differentiation of several independent cell lines obtained by the Type Selection Procedure Cell phones

Grade of Line of Content from Secretion Secretion differentiation cells insulin basal from stimulated (ng / \ mug prot) insulin (pg / \ mug prot) (Pg / \ mug prot) Cell isolated Ix1 0.8 ± 0.1 0.6 ± 0.1 1.1 ± 0.2 without Ix2 0.6 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 differentiation terminal Cell added Ix1 18.2 ± 1.8 83.8 ± 6.1 562.6 ± 47.0 in Ix2 17.0 1.3 76.3 ± 4.3 458.3 ± 29.9 differentiation terminal

As a positive control beta cells were used Adult mouse pancreatic The results obtained were not significantly different from those indicated in the Table one.

4.g) Therapeutic evaluation of beta cells pancreatic differentiated from adult stem cells derived from murine intestinal epithelial cells

Currently under evaluation in vivo therapy in diabetic mice who are going to administer pancreatic beta cells obtained from CMAM for the Cellular Type Selection Procedure provided by the present invention.

Example 5 Obtaining and therapeutic evaluation of pancreatic beta cells differentiated from CMAH derived from a sample of human intestinal epithelial cells 5.a) Cultivation of pluripotential CMAH

Pluripotential CMAH were obtained and cultivated according to the procedure described in Example 3.

5b) -5f)

Proceed in the same way to the procedure described in sections 1.b) -1.f) of Example 1, 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 means of the Procedure of Selection of Cellular Types object of The present invention.

TABLE 4 Insulin content and secretion depending on the differentiation of several independent cell lines obtained by the Type Selection Procedure Cell phones

Grade of Line of Content from Secretion Secretion differentiation cells insulin basal from stimulated (ng / \ mug prot) insulin (pg / \ mug prot) (pg / \ mug prot) Cell isolated MEB1 0.5 ± 0.1 0.4 ± 0.1 0.8 ± 0.1 without MEB2 0.6 ± 0.1 0.5 ± 0.1 0.9 ± 0.2 differentiation MEB3 0.4 ± 0.1 0.4 ± 0.1 0.7 ± 0.1 terminal Cell added MEB1 11.8 ± 1.7 63.5 ± 2.7 421.7 ± 34.7 in MEB2 9.5 ± 1.0 51.1 ± 3.5 342.1 ± 32.4 differentiation MEB3 11.2 ± 1.1 59.8 ± 3.1 395.2 ± 29.9 terminal

As a positive control beta cells were used Adult mouse pancreatic The results obtained were not significantly different from those indicated in Table 1.

5.g) Therapeutic evaluation of beta cells human pancreatic differentiated from CMAH derived from human intestinal epithelial cells

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.

Although the invention has been described by making special reference to the examples presented, anyone familiar with the technique you will quickly appreciate that specific detailed experiments are merely illustrative only of the invention. It should be understood, therefore, that they can be performed one or several modifications to the procedure or cell type precursor or differentiated cell type without leaving the frame described or the basis of the present invention.

Deposit of biological material

On September 23, 1999, it was deposited in the Spanish Type Culture Collection (CECT), Burjasot (Valencia), a culture of Escherichia coli B22, which contains the construction of Figure 1 without the TK gene, to which the deposit number CECT 5197.

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Claims (18)

1. A method for obtaining in vitro stem cells from adult mammalian individuals, including the human species, comprising (i) the gentle disintegration of a tissue sample by using a buffered solution containing a reducing agent and a chelating agent. calcium, (ii) culture, under conditions that keep the disintegrated cells in undifferentiated state, for a period of time sufficient for cell clones to appear, (iii) isolation and growth of the cells obtained, and (iv) verification of their ability to Give rise to tissues. 2. Method according to claim 1, wherein said reducer is dithiothreitol. 3. Method according to claim 1, wherein said calcium chelating agent is ethylenediaminetetraacetate. 4. A stem cell of a mammalian individual adult, including the human species, obtainable according to the method of any of claims 1 to 3. 5. A precursor cell, obtainable according to the method of any of claims 1 to 3, transfected with a segment of DNA that comprises (i) the regions that they contribute to form and stabilize the complex of factors of transcription of a specific gene of a cell type that is specifically expressed in a differentiated cell type, and (ii) a functionally connected selection gene, so that it express when the DNA segment is recognized and transcribed by the complex of transcription factors of the specific gene. 6. Precursor cell according to claim 5, in which said specific gene is a gene that is expressed preferably in a single differentiated cell type. 7. Precursor cell according to claim 5, wherein said specific gene is a gene that is expressed in one or more differentiated cell types. 8. Precursor cell according to claim 5, wherein said specific gene is a gene of human origin and is select among the insulin gene, the rhodopsin gene human, the ornithine transcarbamylase gene and the gene of the albumin. 9. Precursor cell according to claim 5, wherein said selection gene is selected from a gene of resistance, a temperature gene, and their combinations. 10. Precursor cell according to claim 9, wherein said selection gene is selected from the group formed by the gene for resistance to geneticin, the gene of the dihydrofolate resistance, the gene of resistance to puromycin, the interferon gamma resistance gene and the gene of methotrexate resistance. 11. Precursor cell according to claim 1, wherein said segment of DNA further comprises a gene secondary that is expressed in the cell type in which it is expressed the specific gene, functionally connected to a second gene of selection, different from the selection gene connected to the gene specific. 12. Precursor cell according to claim 11, wherein said segment of DNA further comprises the region that contributes to form and stabilize the complex of factors of transcription of said secondary cell type gene to select. 13. Precursor cell according to claim 11, wherein said secondary gene of the cell type to be isolated is select from the group formed by the glucokinase gene, the gene of the glucose transporter Glut-2, the gene of the phosphoenolpyruvate carboxykinase and the albumin gene. 14. Precursor cell according to claim 1, wherein said segment of DNA further comprises 2 or more genes not  specific to the cell type to be isolated but whose expression combined is specific to the cell type to be isolated, operationally connected to their corresponding regulatory regions, being each of said functionally connected regulatory regions with at least one selection gene. 15. Precursor cell according to claim 1, wherein said segment of DNA further comprises a system Genetic biological safety that allows to eliminate cells manipulated 16. Precursor cell according to claim 15, in which said biological safety genetic system is the gene of thymidine kinase from herpesvirus. 17. A method for maturing in vitro until terminal differentiation to a stem cell obtainable by the method of any one of claims 1 to 3, comprising the addition of the factors necessary to achieve its terminal differentiation. 18. Use of a cell according to any of the claims 4 or 5 to 16, for the study of the processes that lead to differentiation and cell dedifferentiation.