Genetic markers for engraftment of human cardiac ventricular progenitor cells
The present invention provides genetic markers for identifying engraftable human cardiac ventricular progenitor cells. The engraftment markers of the invention include angiogenic markers and extracellular matrix markers. Human ventricular progenitor cells expressing these markers are capable of forming ventricular tissue in vivo that is vascularized and supported by an extracellular matrix. Methods of engrafting human cardiac ventricular progenitor cells by transplanting into a subject progenitor cells that express the engraftment markers are also provided.
1. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising:
subjecting human pluripotent stem cells to activation of Wnt/β-catenin signaling on day 0 of culture, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5 of culture to thereby obtain a culture of cardiac progenitor cells (CPCs) comprising LIFR+Islet 1+human ventricular progenitor cells (HVPs);
isolating the LIFR+Islet 1+HVPs on day 5, day 6 or day 7 of culture; and
detecting expression of at least one engraftment marker on the LIFR+Islet 1+HVPs to thereby detect the engraftable HVPs.
2. The method of claim 1 , wherein the at least one engraftment marker detected is at least one positive angiogenic marker.
3. The method of claim 2 , wherein three or more positive angiogenic markers are detected.
4. The method of claim 2 , wherein the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX.
5. The method of claim 1 , wherein the at least one engraftment marker detected is at least one positive extracellular matrix marker.
6. The method of claim 5 , wherein the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1.
7. The method of claim 1 , wherein the at least one engraftment marker detected is at least one positive angiogenic marker and at least one positive extracellular matrix marker.
8. The method of claim 7 , wherein:
the at least one positive angiogenic marker is selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX; and
the at least one positive extracellular matrix marker is selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4 and FREM1.
9. The method of claim 1 , wherein the at least one engraftment marker detected is at least one negative angiogenic marker.
10. The method of claim 1 , wherein the at least one engraftment marker detected is at least one negative extracellular matrix marker.
11. The method of claim 1 , wherein the at least one engraftment marker detected is at least one negative angiogenic marker and at least one negative extracellular matrix marker.
12. The method of claim 1 , wherein detecting expression of at least one engraftment marker comprises detecting expression of mRNA encoding the at least one engraftment marker in the HVPs.
13. The method of claim 1 , wherein detecting expression of at least one engraftment marker comprises detecting lack of expression of mRNA encoding the at least one engraftment marker in the HVPs.
14. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising:
subjecting human pluripotent stem cells to activation of Wnt/β-catenin signaling on day 0 of culture, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5 of culture to thereby obtain a culture of cardiac progenitor cells (CPCs) comprising LIFR+Islet 1+human ventricular progenitor cells (HVPs);
isolating the LIFR+Islet1+HVPs on day 5, day 6 or day 7 of culture; and
detecting expression of engraftment markers on the LIFR+Islet1+HVPs to thereby detect the engraftable HVPs, wherein the engraftment markers comprise (i) ten or more positive angiogenic markers; or (ii) three or more positive extracellular matrix markers.
15. The method of claim 14 , wherein the engraftment markers comprise ten or more positive extracellular matrix markers.
16. The method of claim 14 , wherein the ten or more positive angiogenic markers are selected from the group consisting of: FGF10, PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, GATA6, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, ACVR1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5, HHEX, and combinations thereof.
17. The method of claim 14 , wherein the three or more positive extracellular matrix markers are selected from the group consisting of: FGF10, SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, BMP4, FBLN7, FBLN2, NDNF, HTRA1, HAPLN1, EMILIN1, SPOCK3, PODNL1, IHH, ACAN, NID2, COL4A6, LAMC1, FMOD, MUC4, EMID1, HMCN1, NID1, VCAN, CILP2, SOD3, ADAMTS3, ZP3, ANGPTL4, CRTAC1, LTBP4, FREM1, and combinations thereof.
18. The method of claim 14 , wherein detecting expression of engraftment markers comprises detecting expression of mRNA encoding the engraftment markers in the HVPs.
19. A method of identifying engraftable human ventricular progenitor cells (HVPs), the method comprising:
subjecting human pluripotent stem cells to activation of Wnt/β-catenin signaling on day 0 of culture, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5 of culture to thereby obtain a culture of cardiac progenitor cells (CPCs) comprising LIFR+Islet 1+human ventricular progenitor cells (HVPs);
isolating the LIFR+Islet1+HVPs on day 5, day 6 or day 7 of culture; and
detecting expression of at least one engraftment marker on the LIFR+Islet1+HVPs to thereby detect the engraftable HVPs, wherein the at least one engraftment marker comprises (i) at least one negative angiogenic marker selected from the group consisting of: ETS1, BAX, XBP1, TDGF1, C5AR1, EPHA1, HS6ST1, SHC1, SP100, JAM3, CASP8, FLT4, SFRP2, HPSE, BAK1, GPX1, VAV3, VAV2, EGF, ADAM15 and AGGF1; or (ii) at least one negative extracellular matrix marker selected from the group consisting of: FKBP1A, CLU, TFP12, PLSCR1, FBLN5, VWA1, ADAMTS16, MMP25, SFRP2 and SOD1.
20. The method of claim 19 , wherein at least one negative angiogenic marker is detected and at least one negative extracellular matrix marker is detected.
21. The method of claim 19 , wherein detecting expression of the at least one negative angiogenic marker or the at least one negative extracellular matrix marker comprises detecting lack of expression of mRNA encoding the at least one negative angiogenic marker or at least one extracellular matrix marker in the HVPs.