FIELD OF THE INVENTION The present invention concerns a method for the isolation of stem cells of a human tissue. The method provides to identify and isolate such cells using, for example, the CD 34 positive membrane protein, the CD 133 positive membrane protein or similar as a specific differentiating marker. The method according to the invention allows to obtain stem cells taken from a human tissue, totipotential, with a minimum level of differentiation.

BACKGROUND OF THE INVENTION The importance of transplanting stem cells to a human tissue is well known, to encourage the regeneration of the cells of said tissue, such as for example epithelial cells, in the case of pathologies which witness a serious deterioration of the transparency or morphology of the cornea, such as post-traumatic or post-inflammatory scars, oedemas caused by endothelial imbalance, keratoconus or otherwise.

For example haematopoietic stem cells have been transplanted into the heart of patients who have suffered a heart attack, with the purpose of regenerating new cardiac muscular tissue, in the necrotic area, to allow the organ to fully restore its functions.

In other cases, on the contrary, stem cells are transplanted into a patient, especially when it is necessary to transplant a tissue and there is a risk of rejection of the tissue transplanted due, for example in the case of a corneal tissue, to an alteration in the functionality of the epithelium and with a deficit of stem cells.

The transplantation of stem cells in general, and corneal

stem cells in particular, is necessary especially in the case of patients who have a deficit of such cells and therefore are no longer able to regenerate the cells of the original tissue.

In the case of an eye, the stem cells are localized in the limbus of the corneal epithelium and, before the corneal transplant, are removed from the healthy eye of the patient or, in the event of a pathology in both eyes, from a person compatible with the patient.

In the state of the art, after they have been removed and grown in vitro, the stem cells are identified, so they can be transplanted to the patient, according to the size of the colonies obtained, called"holoclones", and by the presence of a nuclear protein, P-63, which is used as a differentiating marker (PNAS USA, Vol. 98, Issue 6,3156- 3161-March 13,2001).

This conventional method, however, has the disadvantage that it does not allow to isolate the stem cells in a highly selective fashion, particularly in the case of corneal cells, of the limbus zone of the cornea, from other types of cells present in the same zone, for example already differentiated.

The conventional method has the disadvantage that it isolates a heterogeneous population of stem cells with varying levels of differentiation.

The present Applicant has designed and embodied this invention to overcome the shortcomings of the state of the art and to obtain further advantages.

SUMMARY OF THE INVENTION The invention is set forth and characterized essentially in the main claim, while the dependent claims describe other innovative characteristics of the invention.

The purpose of the invention is to perfect a method to

isolate, in a short time, only and exclusively the stem cells of a human tissue, totipotential and not differentiated.

In accordance with this purpose, the method according to the present invention for the isolation of stem cells of a human tissue, such as for example that of a cornea, liver, kidney or similar, comprises at least a step to identify said cells, by means of the specific selection of stem cells expressing, as a differentiating marker or antigen, a membrane protein belonging to the differentiation cluster, also known as CD. The membrane protein comprises for example the CD 34 positive membrane protein, the CD 133 positive membrane protein or similar.

By positive membrane protein we mean that this protein is expressed on the plasmatic membrane of the stem cell.

In this case, the sample of human tissue is removed from a healthy portion of the patient's organ, or from an organ of a person who is compatible with the patient. In a subsequent step, the human tissue is subjected to an enzymatic treatment to obtain the separation of the individual cells which make up the original tissue.

According to a preferential embodiment, after separating the cells from the original tissue and expanding them in vitro, the stem cells are identified and isolated by determining the presence of the CD 34 positive membrane protein (s) and/or CD 133 positive protein (s), or similar.

In the step wherein the stem cells are identified, the method provides to use any conventional procedure already used in the field of isolating cells generating the non- differentiated human haematopoietic line, in order to identify cells which express the CD 34 positive protein, CD 133 positive or similar.

One of the procedures which is used is supplied, for

example, by the Company Miltenyi Biotec. by means of the "MACS"cell isolation kit n. 467-02; it provides a magnetic type separation of the stem cells expressing the CD 34 positive membrane protein. This procedure has been suitably adapted to be able to isolate stem cells of a human tissue, which are different from the blood stem cells.

The method according to the present invention has the advantage that it allows a highly specific and selective isolation of cells with a stem origin of a human tissue expressing the CD 34 positive protein, CD 133 positive or similar, and allows them to be purified from the other types of cell present in the original tissue.

Moreover, the method provides to isolate, quickly and selectively, stem cells of the original tissue and to cultivate them in vitro in a short time, preventing the occurrence of a high differentiation of the cells themselves.

According to another characteristic of the present invention, the method allows to use very small quantities of tissue taken from the original organ, and in any case to obtain a sufficient quantity of stem cells to be transplanted, with a high level of certainty of their identity.

According to another characteristic, the method according to the present invention allows to isolate stem cells of the tissue type and expressing the CD 34 positive membrane protein, the CD 133 positive protein or similar, by means of which it is possible to generate new human tissue and able not only to auto-differentiate itself in the original tissue, but also to restore or even replace that which is not functioning.

BRIEF DESCRIPTION OF THE DRAWINGS These and other characteristics of the present invention

will be apparent from the following description of a preferential form of embodiment, given as a non-restrictive example, with reference to the attached drawings wherein: - fig. 1 is a figurative representation of a part of the method for the isolation of stem cells of a tissue according to the present invention; - fig. 2 is a schematic representation of a stem cell expressing a CD 34 positive membrane protein; - fig. 3a is a schematic representation of a first part of the purification step of stem cells; fig. 3bis a schematic representation of the second part of the purification step of stem cells; -fig. 3c is a schematic representation of the third part of the purification step of stem cells.

DETAILED DESCRIPTION OF A PREFERENTIAL EMBODIMENT With reference to the attached drawings, a method according to the present invention for the isolation of stem cells of a human tissue, such as for example a corneal tissue, is performed in a totally sterile environment and provides, as we have seen, to use an isolation procedure which derives from a conventional procedure, for example the already mentioned Miltenyi Biotec. procedure, which until now has been used only to isolate blood stem cells. This procedure has been modified and adapted for the selective isolation of stem cells, generally of human tissue and specifically of the corneal tissue.

In the example shown here, the method comprises a first step to take a sample 10 of human tissue, such as the basal layer of the limbus of a corneal epithelium. The sample 10, shown schematically in fig. 1, has a surface size in the range of around 1 mm2.

In a second step, the sample 10 is cut and subjected to an enzyme treatment of digestion with collagenase trypsin, CTC,

at a temperature of between 33°C-40°Cfor 10-40 minutes, with the purpose of obtaining a mixed population of individual cells 14, among which there are the stem cells 16 expressing the CD 34 membrane protein.

The mixed population of cells 14 and stem cells 16 is washed in PBS (isotonic phosphate buffer), to eliminate the residual enzyme and subsequently centrifuged.

The cells 14 and stem cells 16 are subsequently dished in a corresponding culture dish 11, where an HECM culture medium 12 is prepared, of a conventional type, comprising a mixture of HUMED and 199 in percentages varying between 40% and 60% ["Biochemical and Biophysical Research Communications, 219,412-417 (1996) and"Biochemie, 82, 1107-1114 (2000)"].

Subsequently, the cells 14 and stem cells 16 are collected with the culture medium 12 and subjected to relative centrifugation for 4. 6 minutes at 1000 rpm. The centrifuged cells are again suspended in 1. 5-3 ml of PBS buffer, containing EDTA 2mM (ethylenediaminotetracetic acid) and 0. 2-0. 7% of BSA (bovine serum albumin).

The cells 14 and stem cells 16 are counted and again subjected to centrifugation for 4-6 minutes at 1000 rpm.

From this centrifugation we obtain a pellet 15 which is again suspended in 300 ll of PBS. The mixed population of cells 14 and 16 thus obtained comprises a number of cells of between about 105 and 108 units.

Subsequently, 100Ll of FcR Blocking reagent 27, that is, the blocking reagent of the receptor for the Fc fragment, is added to the cellular suspension, and after 1. 5 minutes, about 100, ut of CD 34 microbeads 26.

The CD 34 microbeads 26 are able to bind specifically with the CD 34 positive membrane protein expressed only on the corneal stem cells 16 and also comprise a component 26a with

properties of a magnetic type. Fig. 2 shows figuratively the specific coupling respectively between the CD 34 membrane protein and the CD 34 microbeads 26.

The suspension comprising the cells 14 of the original tissue, the stem cells 16 bound to the CD 34 microbeads 26 and the blocking reagent of the receptor for the Fc fragment, is stirred and incubated subsequently for 30 minutes at 4°C. After incubation, another 2ml of PBS is added to the mixture which is subjected to further centrifugation at 1000 rpm for 4. 6 minutes.

The supernatant is removed by suction and the pellet, precipitated, is again suspended in 500/10001 of PBS.

Subsequently, the suspension obtained is filtered with a 30ßm filter to obtain a unicellular suspension 28, that is, not including aggregates but only individual cells.

In the fourth step of real purification of the stem cells 16, the unicellular suspension 28 (fig. 3a) is eluted in a separation column 29 able to be inserted in a support 30, inside which the expansions of a magnet 31 are arranged. The column 29, inserted in the support 30, is subjected to a relative magnetic field.

Subsequently the stem cells 16 are separated (fig. 3b) from the other cells 14 by means of a magnetic-type separation, through the magnetic interaction of the magnetic component 26a of the CD 34 microbeads with the solid part 32 of the column 29.

A first eluate 35, obtained by percolating the unicellular solution 28 in the column 29, contains only CD 34 negative cells 14 on which the CD 34 membrane protein is not present.

The column 29 is subsequently washed with at least 500p1 of PBS.

Subsequently the column 29 is removed from the magnetic support and in turn washed with PBS. In this way the stem

cells 16, bound to the CD 34 microbeads, are released from the solid part 32 (fig. 3c) because, since there is no magnetic field, the CD 34 microbeads 26a no longer interact with the column 29.

The second eluate 36, containing the CD 34 positive cells, is collected in a single suspension, which is subsequently centrifuged for 4. 6 minutes at 1000 rpm. The pellet obtained is collected and transplanted into a patient.

The time duration of the method as described heretofore substantially depends on the quantity of stem cells present in the original sample 10 before the treatment.

It is clear, however, that modifications and/or additions of parts may be made to the method for the isolation and identification of stem cells of a human tissue as described heretofore, without departing from the field and scope of the present invention.

In fact, the method according to the present invention provides to isolate and cultivate the stem cells of a human tissue also using other conventional procedures for the isolation of stem cells expressing the CD 34 positive membrane protein, CD 133 positive and similar.

It is also clear that the method as described heretofore can also be applied not only to cells expressing the CD 34 positive membrane protein, but also to stem cells expressing the CD 133 membrane protein and similar.

It is also clear that the method as described heretofore can also be used to expand in vitro and identify stem cells not only of a corneal tissue but also of other types of human tissue, such as the liver, the kidneys and similar.