WO2009034436A2

WO2009034436A2 - 'hematopoietic cells that express tmcc-1'

Info

Publication number: WO2009034436A2
Application number: PCT/IB2008/002328
Authority: WO
Inventors: Sergio Abrignani; Mariacristina Crosti; Monica Moro
Original assignee: Istituto Nazionale Di Genetica Molecolare-Ingm
Priority date: 2007-09-11
Filing date: 2008-09-09
Publication date: 2009-03-19
Also published as: ITMI20071744A1; WO2009034436A3

Abstract

The present invention concerns ex vivo cells belonging to the hematopoietic system characterized by the presence of the TMCC1 protein on the cell surface, methods to isolate them and use of the cells themselves.

Description

"HEMATOPOIETIC CELLS THAT EXPRESS TMCC l"

DESCRIPTION

The present invention relates to ex vivo cells belonging to the hematopoietic system characterized by the presence of the TMCC-I protein on the cell surface, methods to isolate them and the use of the cells themselves.

The TMCC-I /tmcc-1 gene is known in the art for its genetic sequence. The gene for Homo sapiens is represented by GeneID 460688 in the Entrez gene database denomination (http://www.ncbi.nlm.nih.gov/entrez). The TMCC-I gene is found on chromosome 3. The acronym of the tmcc-1 /TMCC-I protein stands for

Transmembrane and coiled-coil domains protein 1.

The TMCCl gene has been described in a variety of micro-array results for various diseases, including male infertility, VII cranial nerve paralysis and several types of tumors.

The need was felt in the art to improve isolation and recognition procedures for specific cells belonging to the hematopoietic system and to improve their application in the therapeutic/diagnostic/prognostic field.

Within the context of the present invention, the "hematopoietic system" is defined as a set of cells found in a mammal which evolve starting from the hematopoietic stem cell (HSC) found in bone marrow, and develop, according to a dendogramatic lineage, into fully differentiated peripheral blood cells.

Within the context of this invention, "lymphocytes" are defined as a set of cells found in a mammal which evolve starting from lymphoid progenitor cell (LPC) found in bone marrow, and develop according to a dendogramatic lineage into fully differentiated peripheral blood lymphocytes.

The need was also felt in the art to define the metabolic and/or physiological status of a cell belonging to the hematopoietic system. ,

The Applicant has surprisingly found that it is possible to meet the above needs using the presence of the TMCC-I protein on the surface of specific cells belonging to the hematopoietic, preferably the lymphocytic, system. This invention is hereby further illustrated by use of the enclosed Figures. Figure 1 shows the results of an experiment where the distribution of TMCC-I on the surface of peripheral blood lymphocytes is detected by "Fluorescent- activated cell sorting" (FACS) (ref. Example 1).

Figure Ia shows the distribution of TMCC-I on the surface of lymphocytes detected using FACS. The lymphocytes are identified on the basis of standard physical size (Forward Scatter, FSC) and granulosity (Side Scatter, SSC) parameters. The number represents the percentage of lymphocytes expressing TMCC-I.

Figure Ib shows the result of a FACS experiment on peripheral blood mononuclear cells (PBMC), where the upper right quadrant shows the percentage of T-lymphocytes expressing TMCC-I vs the total PBMC population.

Figure Ic shows the result of a FACS experiment on PBMC, where the upper right quadrant shows the percentage of B-lymphocytes expressing TMCC-I vs the total PBMC population.

Figure Id shows the result of a FACS experiment on PBMC₅ where the upper right quadrant shows the percentage of NK cells expressing TMCC-I vs the total PBMC population.

Figure Ie shows the result of a RT-PCR experiment highlighting the significant TMCC-I expression in PBMC cells and in specific lymphocytes. Beta-actin expression was chosen as an experimental control.

Figure 2 shows the results of an experiment (ref. Example 2) showing the different expression of TMCC-I in 1:50 diluted peripheral blood lymphocytes from 5 donors, before and after mitogenic activation with Phytohemagglutinin-L

(PHA).

Figure 2a shows TMCC-I expression on lymphocytes surface as detected using

FACS according to size (Forward Scatter, FSC) and granulosity (Side Scatter,

SSC) parameters for samples from 3 donors. The number represents the percentage of lymphocytes expressing TMCC-I.

Figure. 2b shows TMCC-I expression on the lymphocyte surface as detected using FACS according to size (Forward Scatter, FSC) and granulosity (Side

Scatter, SSC) parameters for samples from 2 donors. It should be noted that the entire, or essentially the entire, cell population does express TMCC-I.

Figure 2c shows TMCC-I expression on the lymphocyte surface after PHA activation, as detected using FACS according to size (Forward Scatter, FSC) and granulosity (Side Scatter, SSC) parameters for samples from 5 donors. It should be noted that the entire, or essentially the entire, population does express

TMCC-I.

Figure 3 shows the results of an experiment (ref. Example 2) indicating the different expression of TMCC-I in 1:150 diluted peripheral blood lymphocytes from 3 donors before and after PHA activation.

Figure 3 a shows the average TMCC-I expression on lymphocyte surface as detected by FACS in samples from 3 donors. The lymphocytes are identified according to physical size (Forward Scatter, FSC) and granulosity (Side Scatter, SSC) parameters. The number represents the percentage of lymphocytes expressing TMCC-I.

Figure 3b shows the average TMCC-I expression on lymphocyte surface as detected by FACS after PHA activation (ref. Example 2) in samples from 3 donors. Lymphocytes are identified according to standard physical size

(Forward Scatter, FSC) and granulosity (Side Scatter, SSC) parameters. The number represents the percentage of lymphocytes expressing TMCC-I.

Figure 4 shows the TMCC-I expression on HSC surface from two separate donors (ref. Example 3). HSCs are identified by FACS according to the expression of markers CD34 and CD45. It should be noted that the entire, or essentially the entire, cell population does expressTMCC-1 in the HSCs from the two donors.

Therefore the scope of the present invention relates to ex vivo cells belonging to the hematopoietic system that are characterized by the presence of the TMCC-I protein on the cells' surface.

In the context of the present invention, "cells" are defined as a number of cells consisting of one or more cells.

In the context of the present invention, a protein expression on a "cell surface" is defined as the expression of a protein which crosses or is anchored to the cell membrane and that shows at least one part of its tri-dimensional structure on the outer surface of the cell membrane.

The cells according to the invention shall preferably comprise hematopoietic stem cells (HSC), common lymphoid or myeloid progenitor cells, proerythroblasts, erythroblasts, myeloblasts, lymphoblasts, monoblasts and mature leukocytes. Mature leucocytes according to the invention shall preferably comprise monocytes, B-lymphocytes, T-lymphocytes and granulocytes. These cells are well known in the art for being constituents of the hematopoietic system and they are distinguished according to methods that are well known in the art for their morphological, genomic and proteomic characteristics. In the context of the present invention the various cell populations within the hematopoietic system are distinguished, as described above and well-known in the art, into what are known as "subpopulations".

In the context of the present invention the term "cells" includes all maturation stages the same cell may go through. For example, the term "B-lymphocytes" includes all the possible phases of a B-lymphocyte from pro-B cells (CD34⁺CD19⁺CD20^"Ig^") to, for example, a plasma cell (CD38⁺CD27⁺CD19^+/" CD20ΗLA-DR^').

According to the invention, the phases of development based on the cells' hematopoietic lineage may be dictated by the cell's position within the organs and within the hematopoietic system vessels. The organs and the hematopoietic system vessels are well known in the art and include the bone marrow, lymph nodes and blood or lymphoid vessels. The Applicant has surprisingly found that the cells according to the invention are present in the various districts in the blood system. Therefore, in the context of the present invention, the cells according to the invention may derive from one or more hematopoietic systems with different or equal districts in the hematopoietic system. The cells according to the invention preferably derive from humans, preferably adults. In the context of this invention, "original cells [specific name]" (such as "original HSC") are defined as the group of cells, whichever they may be, present in vivo or ex vivo without being selected for the presence of TMCC-I. The original cells within the hematopoietic system include both the cells belonging to the hematopoietic system expressing TMCC-I and those that do not express TMCC-I on the surface. Said cells may derive from any cell source belonging to the hematopoietic system well-known in the art, preferably from an in vivo source. Said source is preferably the peripheral blood or the umbilical cord blood.

In one aspect of the invention, the cells according to the invention comprise ^• HSCs. Preferably, the HSCs present in the cells according to the invention shall preferably be from 80 to 100%, more preferably from 90 to 100% and still more preferably 95 to 100% of the original HSCs. It is further more preferred that the HSCs are defined CD34⁺CD45^dim expressing cells (*^m means an intermediate level of expression, within the context of this invention).

In one aspect of the invention, the cells belonging to the hematopoietic system comprise cells belonging to the lymphocytic system. In said aspect of the invention, the cells belonging to the lymphocytic system according to the invention are from 0.1 to 20%, preferably from 1 to 15% and still more preferably from 7 to 10% of cells belonging to the original lymphocytic system. The cells according to the invention shall preferably comprise mature lymphocytes. Mature lymphocytes, within the context of this invention, are B- lymphocytes, T-lymphocytes and NK cells preferably expressing CD 19, CD3 and CD56 respectively.

T-lymphocytes comprised in the cells according to the invention shall preferably be from:

- 2 to 15%, preferably 5 to 7.5% and still more preferably 6 to 7% of the original T-lymphocytes, and/or

- 3 to 6%, preferably 4 to 5%.of the total PBMC population. B-lymphocytes comprised in the cells according to the invention shall preferably be from:

- 2 to 20%, preferably 6 to 10% and still more preferably 8 to 8.5% of the original B-lymphocytes, and/or

- 0.1 to 2%, preferably 0.5 to 0.9%.of the total PBMC population

NK cells comprised in the cells according to the invention shall preferably be from:

- 2 to 20%, preferably 8 to 15% still more preferably 11 to 11.5% of the original NK cells, and/or

- 0.1 to 2%, preferably 0.5 to 0.9%. of the total PBMC population. Another object of the present invention is a method for selecting (identifying and/or isolating) lymphocytic cells according to the invention, characterized by at least a step where the presence of TMCC-I on the surface of such cells is used. A preferred embodiment shall comprise one step before, during or after the step itself where TMCC-I is used, where cells are selected positively or negatively for one or more specific cell subpopulations belonging to the hematopoietic system, such as HSCs. In a preferred embodiment, cells are selected positively or negatively for one or more specific cell subpopulations belonging to the lymphocytic system, such as B- or T-lymphocytes.

In the method for identifying and/or isolating cells, according to the invention, a ligand for TMCC-I, more preferably a proteic ligand, such as an antibody or a lectin protein is preferably used. Therefore, a further object of the present invention is an ex vivo ligand specific for TMCC-I, preferably a polyclonal or monoclonal antibody against TMCC-I. Amongst said ligands, the monoclonal antibody against TMCC-I is preferred. The monoclonal antibody can be produced with well-known methods in the art, such as recombination methods or a method using the Kohler & Midstein technology. Said method shall preferably comprise the following stages: i) Immunize an animal with spleen using the TMCC-I protein in order to induce an immune response, preferably in association with an adjuvant; ii) Remove the animal's spleen and treat so. as to obtain an intact cell suspension and isolate the leucocytes from it, for example B- lymphocytes; iii) Create an hybridoma, for example by means of fusion, from a leukocyte cell isolated from the suspension and resulting in - (ii) with an immortalized cell, such as for example cells from a HGRP^v"mieloma lineage. iv) Enrich the number of cells formed in (iii) through a suitable method, such as a cell feeder layer; v) Select, by means of a negative selection method, cells that have formed a functioning hybridoma, such as growing the cells formed in (iii) on a HAT medium, if a HGRF^A mieloma is used; vi) Isolate cells producing anti TMCC-I antibodies by means of well-known methods in the art such as using a marker-bound TMCC-I, for example a probe; vii) Isolate and multiply the selected cells to produce anti TMCC-I monoclonal antibodies . Said ligands may be used in separation protocols well-known in the art, such as magnetic separation. According to the invention, the method for selecting cells or specific cell subpopulations may comprise both positive and/or negative selection protocols well-known in the art.

A preferred protocol to be used, in the identification and/or isolation of said cell subpopulation, is a flow cytometry protocol capable of isolating the cell subpopulation according to the invention, differentiating between TMCC-I expressing and non-expressing cells. An identification and/or isolation protocol using flow cytometry with fluorochromes (F ACS^® by Beckton-Dickinson) shall be still more preferred, preferably as a final and/or subsequent phase following an enrichment protocol, such as a protocol that comprises the use of specific antibody-bound magnetic spheres.

Example 1 gives a detailed description of an embodiment, as a no-limiting example, of a method for identifying various subpopulations belonging to the hematopoietic cell system according to the invention starting from the blood of a human adult.

A further object of the present invention is the use of the cells according to the invention as described below. In a first embodiment, the cells according to the invention can be used in vitro to assess the effect of compounds or factors on the growth and development of said cells. The compounds can be new or known proteins or other types of molecules derived from humans. The factors can be new mediums used to grow or maintain cells and fluids used in the preparation suitable. Said in viti'o use is preferred if the cells according to the invention are one or more specific subpopulations of the hematopoietic system, preferably subpopulations belonging to the lymphocytic system, hi another embodiment, the cells according to the invention can be used in vitro for diagnostic o prognostic purposes. Mediums and cultures well-known in the art can be used to keep the hematopoietic system cells functioning and said subpopulation is submitted to a diagnostic or prognostic test. The diagnostic or prognostic results are preferably correlated to the presence of TMCC-I on the surface of the cells that have been examined. In a preferred embodiment of said use, the cells undergoing the diagnostic or prognostic test are cells belonging to the lymphocytic system.

Said uses of the cells according to the invention are based on the advantageous fact that the presence of TMCC-I varies according to the cells' metabolic status. When division is induced mitogenically, the presence of TMCC-I on cell surface, according to the invention, increases preferably from 150% to 1000%, more preferably from 200 to 600%, and still more preferably from 230 to 270%. Ex vzvo TMCC-I expressing cells can be used to assess the metabolic status of the cells belonging to the hematopoietic system and preferably the TMCC-I expressing cell system. Said use provides that the number of TMCC-I expressing cells and/or the quantity of TMCC-I expressed on each cell can indicate a metabolic status of the cells belonging to the hematopoietic system, preferably the status of TMCC-I expressing cells. This metabolic status can be linked to the cell metabolism of at least one of the following compounds: carbohydrates, polysaccharides, nucleotides, amino acids, lipids, co-factors and vitamins, secondary metabolites, ATP. This embodiment can be applied to the cells belonging to the entire hematopoietic system or to only one or more specific cell subpopulations such as the lymphocytic system or only B- lymphocytes. The number of TMCC-I" expressing cells and/or the quantity of TMCC-I expressed on each cell can be assessed by means of well-known methods in the art such as a ligand as described above. Amongst said ligands, a monoclonal antibody shall be preferred, even more preferably with a probe linked to it, such as a secondary antibody, in order to measure the number of antibodies present and bound to the cell.

In another embodiment, the presence of TMCC-I on the cells' surface can be used to identify the course and the outcome through the lymphocytic lineage of said cells, and/ore to isolate them. Said course through the lymphocytic lineage depends on the examined cell type and therefore it is linked to its outcome. Lymphocyte lineages can be divided into lineages to produce dendritic cells or lymphoblasts, which subsequently produce mature lymphocytes as previously mentioned.

Furthermore, the presence of TMCC-I on the cells' surface makes it possible to determine the lineage the cell is predetermined for, even before the cell starts to undergo through said lineage. This is particularly beneficial for cells, according to the invention, comprising HSCs or CLPs. From said HSC one can determine, based on the presence of TMCC-I on the cell surface, if the cell will become a common lymphoid or myeloid progenitor cell even before the HSC undergoes such a development. From said CLP one can determine, based on the presence of TMCC-I on the cell surface, if the cell will become a B-lymphocyte, T- lymphocyte, NK cell or a dendritic cell, even before the CLP undergoes said development.

The presence of TMCC-I on the cells' surface is preferably assayed by means of a ligand as described above. Amongst said ligands, a monoclonal antibody is preferred, and even more preferred is a monoclonal antibody linked to a marker,

π such as a secondary antibody, in order to measure the number of antibodies present and bound to the cell.

In another embodiment, the cells according to the invention can be used as a medicament.

In a preferred embodiment of said use as a medicament, a subpopulation of cells belonging to the hematopoietic system according to the invention comprising

HSCs, preferably a HSC subpopulation as mentioned above, is used to prepare a medicament for the prophylaxis, interruption or treatment of diseases showing a non-functioning or "wild-type" TMCC-I. When preparing such a medicament, aspects relating to cell transfusion are taken into account, such as the autologous nature of cells (defined as people with cells having identical HLAs, "Human

Leukocyte Antigen") of the donor's cells vs the recipient's.

The advantage of using the cells according to the invention is that these cells are mitogenically active, therefore prone to dividing when they are transfused.

In a preferred embodiment of said use as a medicament, a cell subpopulation belonging to the hematopoietic system according to the invention, preferably comprising HSCs and/or CLPs and/or lymphoblasts, shall be used to prepare a medicament for diseases or conditions requiring the recovery or the increase in number of cells belonging to the hematopoietic system, preferably the lymphocytic system. An example of said conditions requiring the recovery or the increase in number of cells belonging to the hematopoietic system is the condition characterizing post lympho-ablative treatments, such as radiotherapy resulting from a disease such as leukemia.

Said medicament for recovering or increasing the number of cells belonging to the hematopoietic - preferably the lymphocytic - system is prepared in such a way that it is administered according to methods well-known in the art for the transfusion of cells in a patient. The methods of administration of said medicaments are well known in the art and preferably involve intra-venous and direct injections into bone-marrow. Medicaments prepared according to the invention may also comprise excipients and/or stabilizers and/or delivery vehicles.

The presence of TMCC-I on the cell surface is preferably assayed through a ligand as previously described. Amongst said ligands, a monoclonal antibody is preferable and a monoclonal antibody linked with a marker, for example through a secondary antibody, is even more preferred in order to measure the number of antibodies that are present and bound to the cell. The ex vivo ligand for protein TMCC-I and its possible uses are further objects of the invention. The ligand according to the invention shall preferably be proteic and still more preferably an antibody or lectin protein against TMCC-I. Said antibody shall be monoclonal or multiclonal, preferably monoclonal. Said antibody can be synthesized according . to methods well-known in art as previously described.

The ligand according to the invention shall preferably be present in a composition. Said composition preferably comprises excipients and/or adjuvants and/or stabilizers and /or delivery vehicles and its formulation can be provided through methods well-known in the art. The choice of said excipients and/or adjuvants and/or stabilizers and /or delivery vehicles in the composition varies according to their use, with the proviso that the ability of said ligand is ensured. The ligand according to the invention can be used as a medicament. Said ligand can preferably be used for the preparation of a medicament to be used in a diagnostic or prognostic assay directed to evaluating physiological or molecular aspects involving the cell sub-population that belongs to the lymphocytic system and expresses TMCC-I. Said diagnostic assay can be ex vivo or in vivo. In a preferred embodiment, said ligand is linked to a marker, such as a secondary antibody associated with a probe, such as a secondary antibody-bound fluorescent, phosphorescent or radioactive probe.

In another preferred embodiment, the ligand according to the invention may be used for the preparation of a medicament to modulate the movement of cells according to the invention through the human body. Said cell movement may be modulated under normal physiological conditions or may be caused by an immune response.

In another embodiment, the ligand according to the invention may be used for the preparation of a medicament to qualitatively or quantitative assay the metabolic status, as previously described, of cells belonging to the hematopoietic system. Said assessment of the metabolic status of cells belonging to the hematopoietic system may be carried out either ex vivo or in vivo. In another embodiment, said ligand according to the invention may be used for the preparation of a medicament for the prophylaxis or the interruption or the treatment of diseases in which cells belonging to the hematopoietic system that express TMCC-I are involved. In said embodiment, the cells involved are preferably mature lymphocytes and therefore the diseases are autoimmune in nature, such as non-Hodgkin lymphoma or Lupus. In said cases, the aim is to eliminate aberrant and harmful lymphocytes. Therefore the use of antibodies, preferably monoclonal antibodies against TMCC-I, is preferred because they can trigger an ADCC or CDC autologous cascade to eliminate recognizing leukocytes. An adjuvant is preferably present in the medicament so at to be able to induce an immune response.

In another embodiment, the ligand for TMCC-I, preferably an antibody, preferably monoclonal, is prepared with a substance linked to it, e.g. through a secondary antibody. Said linked substance is toxic or in any case appropriate for eliminating the ligand's target - the cell expressing TMCC-I on its surface. Said toxic substance can be a toxin or a radioactive atom, for example iodine 131 or an enzyme that can subsequently be involved in a monoclonal therapy system known in the art as ADEPT.

Example 1 - Isolation of TMCC-I expressing cell subpopiilations according to the invention from peripheral blood Isolating mononuclear cells from peripheral blood

1. A 10 mL peripheral blood sample from a healthy donor was diluted in 1:3 phosphate buffered saline solution (PBS).

2. 15 ml FicoU-Hypaque (1,077 g/L density) was introduced in a 50 mL Falcon tube and then 30 mL peripheral blood from a healthy donor was stratified above. The blood was inserted very slowly to avoid disrupting the interface. This operation was repeated until the entire sample was used up.

3. The Falcon tube was subsequently centrifuged at 1600 rpm for 30 minutes at room temperature, with no break. Mononuclear cells (PBMCs) positioned themselves on the Ficoll-Hypaque-plasma interface. Said PBMC ring was collected and transferred into a 50 mL Falcon tube.

4. PBMCs were then washed twice with 50 mL PBS containing 5% normal human serum (NHS) and centrifuged for 10 min at 1200 rpm.

5. The pellet was then washed with 50 mL PBS containing 5% NHS and centrifuged for 10 min at 800 rpm.

6. PBMCs resulting in a pellet at the end of step 5 were re-suspended in 10-30 mL of PBS containing 5% NHS at room temperature.

Isolation of cells according to the invention from PBMC

1. Cells were counted using a Burker chamber and 0.3 to 0.5 xlO⁶ PBMCs per sample were stained.

2. Samples were incubated with PBS containing 50% NHS for 20 minutes at room temperature.

3. Samples were centrifuged for 3 minutes at 1500 rpm and, without washing, they were then incubated for 10 minutes in an ice bath with 1 :50 diluted TMCC- 1 antiserum in 100 microliters of PBS containing 5% NHS.

TMCC-I antiserum was prepared according to methods known in the art by immunizing mice with the primary structure of TMCC-I . Negative control samples were incubated for 10 minutes in ice with a non- immunized mouse antiserum in order to calibrate the stain coloration of the FACS-resulting image.

4. Centrifuged sample cells were washed twice with PBS containing 5% NHS by removing the surnatant after centrifugation for 3 minutes at 1500 rpm and re- suspending with PBS containing 5% NHS.

5. Said re-suspended cells were then incubated again for 10 minutes in an ice bath with Goat-anti-mouse IgG-PE (Southern Biotech^®), a well-known 1:100 diluted fluorochrome phycoerythrin (PE)-bonded "secondary" antibody in 100 microliter PBS containing.5% NHS.

13. The cells were then washed twice with PBS containing 5% NHS, centrifuged for 3 minutes at 1500 rpm and re-suspended with PBS containing 5% NHS.

14. 12 mg of mlgG (mouse immunoglobulines) per sample were added to the re- suspended pellet which was incubated for at least 60 minutes in ice.

15. Cells were incubated for 10 minutes in an ice bath with m-apha- hCD19Cychrome (BD Biosciences^®), a known PE-Cy5 fluorochrome-bound monoclonal antibody, with mouse-anti-hCD3FITC (BD Biosciences), a known fluorochrome fluorescein (FITC)-bound antibody and with mouse-anti- hCD56APC (BD Biosciences^®), a know Allophycocianin fluorochrome-bound monoclonal antibody.

16. The stained cells were then washed (centrifuging them for 3 minutes at 1500 rpm) with PBS containing 10% NHS and re-suspended in 500 microliters for FACSCalibur^® acquisition.

17. The BecktonDickinson-FACS^® unit was operated according to protocols well-known in the art that are mentioned in Current Protocols in Immunology (2001), John Wiley and Sons Inc., Unit 5.4.1-5.4.22 and produced the results that are summarized in Figure 1, Figure Ib-Id.

In order to verify data related to the TMCC-I expression provided by the aforementioned FACS experiment, a control experiment was carried out with RT-PCR for monitoring the TMCC-I gene expression in total peripheral blood mononuclear cells and in T lymphocyte and and B lymphocyte and monocyte sub populations.

To this end, cells purified with Ficoll were used to select total peripheral blood mononuclear cells. B-lymphocytes, T-lymphocytes and monocytes (CD 19, CD3 and CD 14) were selected by means of specific antibodies conjugated with magnetic spheres (Miltenyi Biotech) according to the supplier's enrichment protocol.

RNA was extracted from said groups of selected cells using the Qiagen kit (cat#

74104) according to the supplier's protocol. The cDNA was produced starting from 100 ng RNA, using the RetroScript enzyme (Ambion, cat# 1710), according to the supplier's protocol.

2 μl of cDNA were used for RT-PCR-based analysis through TMCC-I -specific primers. For the beta-actin gene, the RT-PCR was carried out as a positive control since it is well known that beta-actin is a protein that is expressed by every cell. Primers are described in compliance with the international WIPO standard ST.25 and their expression was developed using the Patent-In 3.3 programme. Said sequence description, as indicated above, is annexed to the text of the present invention. The primers are : TMCC-I fw: SEQ ID NO. 1

ΪMCC-i rev: SEQ ID NO. 2

Beta-actin gene fw: SEQ ID NO. 3

Beta-actin gene rev: SEQ ID NO. 4

The entire sequences are fully shown in the annex in compliance with the international WIPO Standard ST.25 and developed using the Patent-In 3.3 programme. Conditions used for RT-PCR with ZMCC-i-specific primers were the following:

cDNA: 2 microlitres

TMCCl fw (10 micromolar): 1 microlitre

TMCCl rev (10 micromolar): 1 microlitre

2X Taq PCR Master Mix (Qiagen, cat #201443): 25 microlitres

Sterile water: Until a 50 microlitre final volume is reached.

PCR thermal cycle conditions:

94°C, 3 min 94°C, 30 seconds 55⁰C₅ 30 seconds > 30 cycles

72⁰C, 30 seconds

72°C, lO min oo, 4°C

The results are shown in Figure Ie, where it is clear that peripheral blood mononuclear cells express TMCC-I gene and specifically in T (CD3) and B

(CD19) lymphocytes and monocytes (CD14). The control expression (gene for beta-actin) is also shown.

Example 2 — Evaluation of the TMCCl expression on mitogen-stinmlated cells

- Peripheral blood mononuclear cells from 8 donors were isolated through Filcoll-based separation as shown in Example 1.

- Steps 1-3 were then performed as shown in Example 1 to isolate cells from PBMC. However, only cells from 5 donors underwent a 1:50 dilution whilst cells from the other 3 donors underwent a 1:150 dilution.

- Cells were then introduced in 96 well cell culture plates with a U-shaped bottom with a 0,5x10 /ml concentration in 200 ml culture substrate containing lmg/mL Phytohemmagglutinin-L (PHA-L, Roche).

- Cells are incubated with the mitogen for 18-20 hours.

- After incubation with mitogen, cells are recovered and stained to assess the presence of TMCC-I as described under items 15-17 of Example 1.

The results are shown in Figures 2 and 3 for 1:50 and 1:150 dilutions respectively. It can be observed that in both dilutions the presence of TMCC-I increases when they are activated mitogenically. Example 3 - Isolation of cell sub-populations according to the invention in umbilical cord blood

The same isolation procedure above described for peripheral blood was repeated to isolate CBMC instead of PBMC. The protocol was repeated for two separate donors.

Isolation of mononuclear cells from umbilical cord blood (CBMQ

One sac of umbilical cord blood (75ml) was obtained from the Milan Cord

Blood Bank and submitted to a 1:3 phosphate buffered saline solution (PBS) containing 2 mM ethylenediamine tetraacetic acid (EDTA).

A Ficoll-Hypaque separation was then performed following the protocol described in Example 1.

Isolation of cells according to the invention from PBMC

Cells were counted using a Burker chamber and 3xl0⁶-5xl0⁶ CBMCs per sample were stained.

The same protocol as that described in Example 1, steps 1-17, was implemented.

The only change was introduced in step 15. At step 15, cells were incubated again for 10 minutes in an ice bath with mouse-anti-hCD34PC5 (Beckman

Coulter^®), a well-known PE-Cy5 fluorochrome-bound monoclonal antibody and with mouse-anti-hCD45FITC (BD Biosciences), a well-known fluorochrome fluorescein (FITC)-bound monoclonal antibody.

The BecktonDickinson-FACS^® unit was operated according to well-known protocols in the art that are mentioned in Current Protocols in Immunology

(2001), John Wiley and Sons Inc., Unit 5.4.1-5.4.22 and produced the results that are summarized in Figure 4.

The fluorescences that are shown indicate that in two separate donors the entire or essentially the entire HSC cell population express TMCC-I.

Claims

1. Ex vivo cells belonging to the hematopoietic system, characterised by the presence of the TMCC-I protein on the cell surface.

2. . The cells according to claim 1, wherein the cells belonging to the hematopoietic system are selected from the group consisting of HSCs, common lymphoid and common myeloid progenitor cells, erythroblasts, myeloblasts, lymphoblasts and leukocytes.

3. The cells according to claim 2, wherein the leukocytes are selected from the group consisting of monocytes, B-lymphocytes, T- lymphocytes and NK cells.

4. The cells according to any of the claims 1 to 3, wherein the hematopoietic stem cells are comprised in. said cells and account from 80 to 100%, more preferably from 90 to 100% and still more preferably from 95 to 100% of original hematopoietic stem cells.

5. The cells according to any of the claims 1 to 3, wherein the cells belonging to the lymphocytic system are comprised in said cells and account from 0.1 to 20%, more preferably 1 to 15%, and still more preferably 7 to 10% of cells belonging to the lymphocytic system.

6. The cells according to claim 5, wherein the cells belonging to the lymphocytic system comprise B-lymphocytes and account from 2 to 20%, more preferably 6 to 10% and still more preferably 8 to 8.5% of original B-lymphocytes.

7. The cells according to claim 5, wherein cells belonging to the lymphocytic system comprise T-lymphocytes and account from 2 to 15%, more preferably 5 to 7.5% and still more preferably 6 to 7% of original T-lymphocytes.

8. The cells according to claim 5, wherein the cells belonging to the lymphocytic system comprise NK cells and account from 2 to 20%, more preferably 8 to 15% and still more preferably 11 to 11.5% of original NK cells.

9. A method to identify and/or isolate cells according to any of the claims from 1 to 8, characterized by at least one step where the presence of TMCC-I on the surface of said cells is used.

10. The method according to claim 9, wherein before, during or after said stage the presence of TMCC-I on cell surface is used, cells are selected by or reduced to one or more specific sub-populations of cells belonging to the lymphocytic system.

11. The method according to claim 9 or 10, wherein said presence of TMCC-I on the cell surface is used by means of a ligand, preferably a monoclonal antibody against TMCC-I .

12. Use of the cells according to one or more claims 1 to 8 to assess in vitro the effect of compounds or factors on the growth or maturation of said cells.

13. Use of the cells according to one or more claims 1 to 8 in an in vitro diagnostic or prognostic assay.

14. The cells according to one or more claims 1 to 8 for use as a medicament.

15. Use of the cells according to one or more claims 1 to 8 that comprises HSCs and/or myeloid progenitor cells and/or lymphoid progenitor cells, for the preparation of a medicament for the repristination of a cell population belonging to a hematopoietic cell lineage.

16. Use of the cells according to one or more claims 1 to 8, which comprises HSCs, for the preparation of a medicament for the treatment of people missing a functioning or "wild-type" TMCC-I gene.

17. Use of the cells according to any claims 15 to 16, wherein the cell population is present in a composition, preferably comprising excipients and/or stabilizers and/or delivery vehicles.

18. Use of the presence of TMCC-I on the surface of ex vivo cells belonging to the hematopoietic system to assess the cells' metabolic status.

19. The use according to claim 18, wherein the presence of TMCC-I on the cell surface is measured using ligands for TMCC-I, preferably monoclonal antibodies against TMCC-I.

20. The use according to claim 18 or 19, wherein the cells belonging to the hematopoietic system are one or more specific cell sub- populations.

21. An ex vivo ligand specific for TMCC- 1.

22. The ligand according to claim 21, wherein the ligand is an antibody or lectin specific for TMCC-I .

23.. The ligand according to claim 22, wherein the ligand is an antibody, preferably a monoclonal antibody. 24. The ligand according to any of the claims 21 to 23 to be used as medicament. preparation of a medicament for a diagnostic or prognostic assay.

26. Use of the ligand according to any of the claims 21 to 23 for the preparation of a medicament to qualitatively or quantitatively assess the metabolic status of cells belonging to the hematopoietic system.

27. Use of the ligand according to any of the claims 21 to 23 for the preparation of a medicament to modulate movement of cells according to one or more claims 1 to 9 throughout the human body.

28. Use of the ligand according to any of the claims 21 to 23 for the preparation of a medicament for the prophylaxis, interruption or treatment of diseases in which cells expressing TMCC-I and belonging to the hematopoietic system are involved.

29. The use according to the claim 28, wherein the cells expressing TMCC-I and belonging to the hematopoietic cells comprise leukocytes.

30. The use according to the claim 29, wherein diseases are autoimmune.

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