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1. (WO2018224983) PROCÉDÉ D'ISOLEMENT DE SOUS-POPULATIONS DE CELLULES PROGÉNITRICES CARDIAQUES ET UTILISATIONS ASSOCIÉES DANS LE DOMAINE MÉDICAL
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CLAIMS

1. Method for the isolation of human cardiac progenitor cells subpopulations, comprising the following steps:

a) mincing a heart tissue sample containing a population of cardiac cells; b) 4-times progressive digestion of the cardiac tissue with an enzymatic mixture until a cell suspension is achieved and filtration is achieved through filters of size comprised between 30 μΜ and 100 μΜ;

c) further digestion of residual cardiac tissue obtained in step b) for 16 hours with an enzymatic mixture until a cell suspension is achieved and filtration through filters of size comprised between 30 μΜ and 100 μΜ;

d) culturing the cell suspension obtained in step b) and c) in a culture medium suitable to propagate the cardiac cells;

e) preliminary expansion of the cell suspension obtained in step d) in a culture medium suitable to expand the cardiac cells in the presence of an enzymatic or non enzymatic solution;

f) secondary expansion of the cardiac cells obtained in step e) in a culture medium suitable to further expand the cardiac cells in the presence of an enzymatic or non enzymatic solution;

g) isolating one or more subpopulations of cardiac cells by positive and/or negative selection by the use of monoclonal antibodies directed against one or more surface antigens expressed in the starting population of cardiac progenitor cells.

2. Method for the isolation of human cardiac progenitor cell subpopulations according to claim 1, wherein said heart tissue sample of step a) is selected from the group consisting of right auricle, left auricle, septum, apex or ventricular biopsy.

3. Method for the isolation of human cardiac progenitor cell subpopulations according to anyone of the claims 1-2, wherein the mincing of step a) occurs in manual or automatic mode.

4. Method for the isolation of human cardiac progenitor cell subpopulations according to anyone of the claims 1-3, wherein said enzymatic mixture of step b) and c) comprises a basal medium and an enzymatic solution.

5. Method for the isolation of human cardiac progenitor cells subpopulations according to claim 4, wherein said enzymatic solution comprises a mixture of collagenases and/or proteases.

6. Method for the isolation of human cardiac progenitor cells subpopulations according to the anyone of the claims 4-5, wherein said coUagenase and/or protease mixture comprises one or more enzymes selected from the group consisting of coUagenase I, coUagenase II, coUagenase IV, trypsin, EDTA, accutase, coUagenase A, dispase and liberase.

7. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 4-6, wherein said basal medium is Ham's/F12.

8. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 4-7, wherein said coUagenase mixture is present in a concentration comprised between 0.1 and 3 mg/ml.

9. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 1-8, wherein said medium suitable for multiplying and expanding the cardiac cells of steps d), e) and f) is Ham's/F12 medium supplemented with 10% fetal bovine serum, 2 mM L-glutathione and 5 x 10" U/ml human erythropoietin, 10 ng/ml of basic fibroblast growth factor and antibiotics.

10. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 1-9, wherein the enzymatic or non enzymatic solution of step e) and f) for cell expansion is selected from the group consisting of Tryple™ Select, a mixture of trypsin and/or EDTA, Cell dissociation buffer, Cell Stripper, accutase, or dispase.

11. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 1- 10, wherein said monoclonal antibodies of step g) are marked with biotin or a fluorescent molecule, preferably selected between FITC or APC.

12. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 1- 11, wherein said antibodies of step g) are monoclonal antibodies against one or more antigens selected from the group consisting of:

CDla CDlb CDlc CDld CD2

CD3 CD4 CD5 CD6 CD7

CD8a CD9 CD10 CDlla CDllb

CDllb activated CDllc CD13 CD14 CD15 (SSEA-1)

CD16 CD18 CD19 CD20 CD21

CD22 CD23 CD24 CD25 CD26

CD27 CD28 CD29 CD30 CD31

CD32 CD33 CD34 CD35 CD36

CD38 CD39 CD40 CD41 CD42b

CD43 CD44 CD45 CD45RA CD45RB

CD45RO CD46 CD47 CD48 CD49a

CD49c CD49d CD49e CD49f CD50(ICAM-3)

CD51 CD51/61 CD52 CD53 CD54

CD55 CD56(NCAM) CD57 CD58 CD59

CD61 CD62E CD62L CD62P(P-Selectin) CD63

CD64 CD66a/c/e CD66b CD69 CD70

CD71 CD73 CD74 CD79b CD80

CD81 CD82 CD83 CD84 CD85a(ILT5)

CD85d(ILT4) CD85g(ILT7) CD85h(ILTl) CD85j (ILT2) CD85k(ILT3)

CD86 CD87 CD88 CD89 CD90 (Thyl)

CD93 CD94 CD95 CD96 CD97

CD99 CD 100 CD101(BB27) CD 102 CD 103

CD 104 CD 105 CD 106 CD107a(LAMP-l) CD 108

CD 109 CD111 CD112(Nectin-2) CD114 CD115

CD116 CD117 (c-kit) CD119(IFN-g R alpha chain) CD122 CD123

CD 124 CD126(IL-6Ralpha) CD127(IL7R alpha) CD129(IL-9 R) CD131

CD132 CD 134 CD135 CD137(4-1BB) CD137L(4-lBBLigand)

CD 138 CD 140a CD 140b CD141 CD 143

CD 143 CD 146 CD 148 CD150(SLAM) CD152

CD 154 CD155(PVR) CD156c(ADAM10) CD158a/h CD158b ( Kl R2DL2/L3, N KAT2)

CD158d CD158el(KIR3DLl,NKBl) CD158F CD161 CD162

CD163 CD 164 CD 165 CD 166 CD167a(DDRl)

CD 169 CD170(Siglec-5) CD172a(SIRPa) CD172b(SIRPg) CD172g(SIRPg)

CD178(Fas-L) CD 179a CD 179b CD180(RP105) CD181(CXCR1)

CD182(CXCR2) CD 183 CD184(CXCR4) CD193(CCR3) CD195(CCR5)

CD 196 CD197 (CCR7) CD200(OX2) CD200 R CD201(EPCR)

CD202b (Tie2/Tek) CD203c(E-NPP3) CD205(DEC-205) CD206(MMR) CD207(Langerin)

CD209 (DC-SIGN) CD210(IL-10R) CD213a2 CD215(IL-15 Ralpha) CD218a(IL-18R alpha)

CD220 CD221(IGF-1R) CD226(DNAM-1) CD229(Ly-9) CD231 (TALLA)

CD235ab CD243 CD244(2B4) CD245(p220/240) CD252(OX40L)

CD253 (Trail) CD254 CD255 (TWEAK) CD257 (BAFF,BLYS) CD258 (LIGHT)

CD261(DR4,TRAIL-R1) CD262(DR5,TRAIL-R2) CD263(DcRl,TRAIL-R3) CD266(Fnl4,TWEAK F CD267(TACI)

CD268 (BAFF-R, BAFFR) CD270(HVEM) CD271 CD273(B7-DC,PD-L2) CD274(B7-H1,PD-L1)

CD275 (B7-H2,B7-RP1,IC0SL) CD276 CD277 CD278(ICOS) CD279(PD-1)

CD282 (TLR2) CD284 (TLR4) CD286 (TLR6) CD290 CD294

CD298 CD300e (IREM-2) CD300F CD301 CD303

CD 304 CD307 CD307d(FcRL4) CD314(NKG2D) CD317

CD318(CDCP1) CD319(CRACC) CD324(E-Cadherin) CD325 CD326(Ep-CAM)

CD328(Siglec-7) CD334(FGFR4) CD335(NKp46) CD336(NKp44) CD337(NKp30)

CD338(ABCG2) CD340(erbB2 HER- 2) CD344(Frizzled-4) CD351 CD352(NTB-A)

CD354(TREM-1) CD355 (CRTAM) CD357(GITR) CD360(IL-21R) beta2-microglobulin

BTLA C3AR C5L2 CCR10 CLEC12A

CLEC9A CX3CR1 CXCR7 delta-Opioid Receptc DLL1

DLL4 DR3 (TRAMP) EGFR erbB3/HER-3 Fee Rial pha

FcRL6 Galectin-9 GARP (LRRC32) HLA-A,B,C HLA-A2

HLA-DQ HLA-DR HLA-E HLA-G IFN-g Rb chain

Ig light chain k Ig light chain omega IgD IgM IL-28RA

Integrin alpha9betal Integrin beta5 Integrin beta7 Jagged 2 LAP

Lymphotoxin beta receptor (1 Mac-2(Galectin-3) MAIR-II MICA/MICB PE MSC(W3D5)

MSC(W5C5) MSC(W7C6) MSCand NPC(W4A5) MSCA-1(MSC,W8B2) NKp80

Notch 1 Notch2 Notch 3 Notch4 NPC(57D2)

Podoplanin Pre-BCR PSMA Siglec-10 Siglec-8

Siglec-9 SSEA-1 SSEA-3 SSEA-4 SSEA-5

TCRg/d TCR Vbetal3.2 TCRVbeta23 TCRVbeta8 TCRVbeta9

TCR Vdelta2 TCR Vg9 TCRValpha24-Jalphal8 TCRValpha7.2 TCR alpha/beta

Tim-1 Tim-3 Tim-4 TLT-2 TRA-1-60R

TRA-1-81 TSLPR(TSLP-R)

13. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the preceding claims wherein the selection of step g) is carried out by immunomagnetic separation.

14. Method for the isolation of human cardiac progenitor cells subpopulations according to anyone of the claims 11- 13, wherein said monoclonal antibodies of step g) are anti-CD 117 and/or anti-CD90.

15. Subpopulation of human cardiac progenitor cells obtained by the method of isolation according to claim 14, characterized by a surface marker profile selected between CD90" and CD117+ +/CD90".

16. Subpopulation of human cardiac progenitor cells according to claim 15, for use in medical field, alone or in combination with another subpopulation of human cardiac progenitor cells.

17. Subpopulation of human cardiac progenitor cells according to claim 16, for use in the cardiovascular cell therapy or cardiac cell and/or tissue transplantation field.