Genetic markers for engraftment of human cardiac ventricular progenitor cells
Inventors
LEUNG, Chuen Yan • Clarke, Jonathan • XU, Jiejia • SANTORO, Federica • SAHARA, Makoto • Chien, Kenneth R.
Assignees
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Publication Number
US-10612094-B2
Publication Date
2020-04-07
Expiration Date
Abstract
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.
Core Innovation
The invention relates to transplanting engraftable human ventricular progenitor cells (HVPs) obtained from human pluripotent stem cells. Human cells containing cardiac progenitor cells (CPCs) are cultured under conditions causing differentiation into human ventricular progenitor cells (HVPs) by activation of Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5, to thereby obtain a culture of day 5-7 CPCs comprising day 5-7 LIFR+ Islet1+ HVPs.
To identify and isolate engraftable HVPs, the patent teaches detecting expression on the day 5-7 HVPs of at least one surface marker selected from JAG1, FZD4, FGFR3 and/or TNFSF9. The patent further teaches detecting expression in the day 5-7 HVPs of at least one engraftment marker, and isolating day 5-7 HVPs that co-express the at least one surface marker and the at least one engraftment marker to thereby isolate engraftable day 5-7 HVPs.
The patent also defines identification strategies for engraftable HVPs using panels of engraftment markers that include positive angiogenesis markers and positive extracellular matrix markers. It identifies engraftable HVPs by detecting expression of at least one positive angiogenesis marker and/or at least one positive extracellular matrix marker in the HVPs, where the HVPs also express at least one surface marker selected from JAG1, FZD4, LIFR and/or TNFSF9. In further examples, the patent specifies thresholds such as three or more positive extracellular matrix markers or ten or more positive angiogenic markers.
Claims Coverage
The independent claims cover four related methods centered on engraftability-focused selection of day 5-7 HVPs. The inventive features include timed Wnt/β-catenin activation and inhibition to obtain day 5-7 LIFR+ Islet1+ HVPs, surface-marker expression criteria, and engraftability-marker detection using specified angiogenic and extracellular-matrix marker sets with positive-marker detection thresholds.
Wnt/β-catenin differentiation to obtain day 5-7 LIFR+ Islet1+ HVPs
Culturing human cells containing cardiac progenitor cells (CPCs) under conditions causing differentiation into human ventricular progenitor cells (HVPs) by subjecting human pluripotent stem cells to activation of Wnt/β-catenin signaling on day 0, followed by inhibition of Wnt/β-catenin signaling from day 3 to day 5 to thereby obtain a culture of day 5-7 CPCs comprising day 5-7 LIFR+ Islet1+ HVPs.
Surface marker detection on day 5-7 HVPs
Detecting expression on the HVPs of at least one surface marker selected from the group consisting of JAG1, FZD4, FGFR3 and/or TNFSF9 on the day 5-7 HVPs.
Engraftment-marker detection and isolation of co-expressing engraftable HVPs
Detecting expression in the day 5-7 HVPs of at least one engraftment marker; isolating day 5-7 HVPs that co-express the at least one surface marker and the at least one engraftment marker to thereby isolate engraftable day 5-7 HVPs.
Transplantation to form a vascularized, electrically responsive ventricular muscle patch
Transplanting the engraftable day 5-7 HVPs into a subject such that the engraftable day 5-7 HVPs form a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix.
Identify engraftable HVPs by three or more positive extracellular matrix markers
Detecting expression of three or more positive extracellular matrix markers 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 in the HVPs to thereby identify engraftable HVPs.
Identify engraftable HVPs by positive angiogenesis markers and/or positive extracellular matrix markers with day 5-7 surface marker expression
Detecting expression of at least one positive angiogenesis marker selected from the group consisting of PRKD1, CCBE1, PDGFRA, EPHB2, GATA2, NTRK1, PTGIS, BMPER, BMP4, C1GALT1, MEIS1, TBX1, PKNOX1, ID1, TCF21, HEY1, HOXB3, HGF, IL6, GHRL, IHH, SRPK2, HAND1, AMOT, NRP2, PTEN, SEMA3E, APOLD1, SETD2, DAB2IP, KDR, PGF, EMP2, TAL1, HIPK2, CSPG4, TNFAIP3, NRP1, NFATC4, CDC42, ANGPTL4, BCAS3, HIPK1, NRXN3, FZD5 and HHEX; and/or detecting expression of at least one positive extracellular matrix marker selected from the group consisting of SMOC1, CCBE1, COL6A6, ADAMTS12, COL19A1, LAMA1, 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 in the HVPs to thereby identify engraftable HVPs, wherein the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs) derived by activation of Wnt/β-catenin signaling on day 0 and inhibition from day 3 to day 5, and wherein the HVPs also express at least one surface marker selected from JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
Identify engraftable HVPs by ten or more positive angiogenic markers
Detecting expression of ten or more positive angiogenic markers in the HVPs to thereby identify engraftable HVPs, wherein the HVPs are obtained from a culture of day 5-7 cardiac progenitor cells (CPCs) derived by activation of Wnt/β-catenin signaling on day 0 and inhibition from day 3 to day 5, and wherein the HVPs also express at least one surface marker selected from JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9.
The independent claims collectively define engraftability-focused selection of day 5-7 HVPs by combining timed Wnt/β-catenin modulation to obtain HVPs, surface-marker expression requirements (JAG1, FZD4, LIFR, FGFR3 and/or TNFSF9), and engraftability-marker detection using specified angiogenic and extracellular-matrix marker sets with positive-marker detection thresholds. The transplanting claim further specifies that the selected engraftable HVPs form a vascularized, electrically responsive ventricular muscle patch secreting extracellular matrix.
Stated Advantages
Allows isolating engraftable day 5-7 HVPs by detecting co-expression of a surface marker and engraftment markers.
After transplantation, engraftable day 5-7 HVPs form a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix.
Documented Applications
Transplantation into a subject to form a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix.
Identification/selection of engraftable HVPs to exclude Isl1-negative (non-engraftable) cells for transplantation.
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