WO2003093457A1 - Method for the identification and isolation of stem cells from a human tissue - Google Patents

Method for the identification and isolation of stem cells from a human tissue Download PDF

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Publication number
WO2003093457A1
WO2003093457A1 PCT/IB2003/001661 IB0301661W WO03093457A1 WO 2003093457 A1 WO2003093457 A1 WO 2003093457A1 IB 0301661 W IB0301661 W IB 0301661W WO 03093457 A1 WO03093457 A1 WO 03093457A1
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Prior art keywords
stem cells
membrane protein
cells
positive
human tissue
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PCT/IB2003/001661
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French (fr)
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Giuseppina Perrella
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Universita' Degli Studi Di Udine
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Priority to AU2003219429A priority Critical patent/AU2003219429A1/en
Publication of WO2003093457A1 publication Critical patent/WO2003093457A1/en

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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • 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 10 method according to the invention allows to obtain stem cells taken from a human tissue, totipotential, with a minimum level of differentiation.
  • transplanting stem cells to a human 15 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, _0 oedemas caused by endothelial imbalance, keratoconus or otherwise.
  • haematopoietic stem cells have been transplanted into the heart of patients who have suffered a heart attack, with the purpose of regenerating new cardiac
  • stem cells are transplanted into a patient, especially when it is necessary to transplant a tissue and there is a risk of rejection of BO 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.
  • stem cells in general, and corneal stem cells in particular are 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.
  • 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.
  • 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 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 .
  • 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.
  • the method according to the present invention for the isolation of stem cells of a human tissue 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.
  • positive membrane protein we mean that this protein is expressed on the plasmatic membrane of the stem cell.
  • 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.
  • the human tissue is subjected to an enzymatic treatment to obtain the separation of the individual cells which make up the original tissue.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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 .
  • 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. 3b is 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.
  • a method according to the present invention for the isolation of stem cells of a human 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.
  • 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 mm 2 .
  • 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.
  • PBS isotonic phosphate buffer
  • 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) "] .
  • 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.
  • the mixed population of cells 14 and 16 thus obtained comprises a number of cells of between about 10 5 and 10 8 units.
  • 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 lOO ⁇ l 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 500/1000 ⁇ l of PBS.
  • the suspension obtained is filtered with a 30 ⁇ m filter to obtain a unicellular suspension 28, that is, not including aggregates but only individual cells.
  • 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.
  • 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 500 ⁇ l of PBS.
  • the column 29 is removed from the magnetic support and in turn washed with PBS.
  • 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.
  • 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.
  • 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.

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Abstract

Method for the identification and isolation of stem cells (16) of a human tissue (10), which provides at least an identification and isolation step, by means of specific selection of stem cells (16) of human tissue (10), expressing as a differentiating marker a membrane protein belonging to the cluster differentiation CD.

Description

METHOD FOR THE IDENTIFICATION AND ISOLATION OF STEM CELLS FROM A HUMAN TISSUE
•k -k "k - -λ-
FIELD OF THE INVENTION 5 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 10 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 15 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, _0 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
_5 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 BO 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. 3b is 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 μl 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, lOOμl 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 lOOμl 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 500/1000μl of PBS.
Subsequently, the suspension obtained is filtered with a 30μm 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 500μl 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.

Claims

1. Method for the identification and isolation of stem cells of a human tissue, characterized in that it provides at least an identification and isolation step, by means of specific selection, of stem cells (16) of human tissue, expressing as a differentiating marker a membrane protein belonging to the cluster differentiation CD.
2. Method for the identification and isolation of stem cells (16) of a tissue of a corneal limbus, characterized in that it provides at least an identification and isolation step, by means of specific selection, of stem cells (16) of human tissue, expressing as a differentiating marker a membrane protein belonging to the cluster differentiation CD.
3. Method as in claim 1 or 2, characterized in that said membrane protein comprises the CD 34 positive membrane protein.
4. Method as in claim 1 or 2 , characterized in that said membrane protein comprises the CD 133 positive membrane protein.
5. Method as in any claim hereinbefore, characterized in that said isolation step comprises a substep of enzymatic treatment of said human tissue (10) by means of collagenase trypsin in order to separate a mixed population of cells (14) of said human tissue (10) , among which there are stem cells (16) of said original tissue (10) expressing the CD 34 membrane protein and/or CD 133 and/or similar.
6. Method as in claim 5, characterized in that it comprises a step of preparing said mixed population of cells (14, 16) during which said mixed population is dished in a determinate culture medium (12) to obtain a cellular suspension comprising a number of said cells (14, 16) of between about 105 and 108 units.
7. Method as in claim 6, characterized in that at least a blocking reagent of the receptor for the Fc fragment (27) is added to said cellular suspension.
8. Method as in claim 6, characterized in that an antibody (26) able to bind specifically with the CD 34 positive and/or CD 133 positive membrane protein and/or similar is added to said cellular suspension.
9. Method as in claim 6, characterized in that an antibody (26) able to bind specifically with the CD 34 positive and/or CD 133 positive membrane protein and/or similar and comprising at least a component (26a) having magnetic type properties is added to said cellular suspension.
10. Method as in claim 9, characterized in that said cellular suspension (28) comprising said antibody (26) is eluted, after having been filtered, in a separation column (29), provided with a solid part (32), able to be subjected to a relative magnetic field, in order to separate said stem cells (16) expressing said CD 34 positive and/or CD 133 protein and/or similar from the other cells (14) , by means of the magnetic interaction of said magnetic component (26a) of said antibody (26) with said solid part (32) of said separation column (29).
11. Method as in claim 9, characterized in that said stem cells (16) are removed from said separation column (29) by cancelling said magnetic field.
PCT/IB2003/001661 2002-04-30 2003-04-23 Method for the identification and isolation of stem cells from a human tissue WO2003093457A1 (en)

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WO2005079145A2 (en) 2004-01-27 2005-09-01 Reliance Life Sciences Pvt. Ltd. Tissue system with undifferentiated stem cells derived from corneal limbus
US8067233B2 (en) 2004-02-26 2011-11-29 Reliance Life Science Pvt. Ltd. Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof
US8187875B2 (en) 2004-02-26 2012-05-29 Reliance Life Sciences Pvt. Ltd. Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
US8338175B2 (en) 2006-02-24 2012-12-25 Reliance Life Sciences Pvt. Ltd. Conjunctival tissue system

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D. DONNINI ET AL.: "A new model of human aortic endothelial cells in vitro", BIOCHIMIE, vol. 82, 2000, MASSON, PARIS, FR, pages 1107 - 1114, XP002232664, ISSN: 0300-9084 *
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DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 16 November 2001 (2001-11-16), GORDON PAUL R ET AL: "Large scale isolation of CD133+ progenitor cells from GCSF mobilized peripheral blood stem cells.", XP002232665, Database accession no. PREV200200220375 *

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US6388132B1 (en) 1995-03-10 2002-05-14 G. D. Searle & Co. Bis-amino acid hydroxyethylamino sulfonamide retroviral protease inhibitors
WO2005079145A2 (en) 2004-01-27 2005-09-01 Reliance Life Sciences Pvt. Ltd. Tissue system with undifferentiated stem cells derived from corneal limbus
US8067233B2 (en) 2004-02-26 2011-11-29 Reliance Life Science Pvt. Ltd. Pluripotent embryonic-like stem cells derived from corneal limbus, methods of isolation and uses thereof
US8187875B2 (en) 2004-02-26 2012-05-29 Reliance Life Sciences Pvt. Ltd. Dopaminergic neurons derived from corneal limbus, methods of isolation and uses thereof
US8338175B2 (en) 2006-02-24 2012-12-25 Reliance Life Sciences Pvt. Ltd. Conjunctival tissue system

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