Use of Neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells
Inventors
Chien, Kenneth R. • Clarke, Jonathan • LEUNG, Chuen Yan
Assignees
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Publication Number
US-11186820-B2
Publication Date
2021-11-30
Expiration Date
Abstract
The present invention provides NRP1 as a cell surface marker for isolating human cardiomyogenic ventricular progenitor cells (HVPs), in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Additional HVP cell surface markers identified by single cell sequencing are also provided. The invention provides in vitro methods of the separation of NRP1+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated NRP1+ ventricular progenitor cells are also provided. Methods of in vivo use of NRP1+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the NRP1+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.
Core Innovation
The invention relates to a method for isolating human cardiac ventricular progenitor cells. The method obtains a culture of human cells containing cardiac progenitor cells by subjecting human pluripotent stem cells to Wnt/β-catenin signaling activation on day 0 of culture and inhibiting Wnt/β-catenin signaling activation on day 3 to day 5 of culture. The resulting culture is then contacted with one or more agents reactive with neuropilin-1 (NRP1).
After contact with the one or more agents reactive with NRP1 on day 5-7 of culture, NRP1 reactive positive cells are separated from non-reactive cells. This separation thereby isolates NRP1+ human cardiac ventricular progenitor cells. The method is based on using NRP1 as the cell-surface marker through NRP1-reactive agents during the defined culture window.
The disclosed NRP1-reactive agent includes an anti-NRP1 antibody or a soluble NRP1 ligand, including VEGF-A or Sema3A. The document also describes gene-expression profiling of human ventricular progenitor cells, including extracellular matrix gene sets associated with Isl1 expression, and reports NRP1 selection based on co-clustering and association with high Isl1 expression. NRP1+ HVPs are further characterized by flow cytometry with coexpression of TRA-1-60 and ISL1.
Claims Coverage
The claim coverage centers on one method for isolating NRP1+ human cardiac ventricular progenitor cells, combining timed Wnt/β-catenin modulation, NRP1-reactive contacting, and separation of NRP1 reactive positive cells. Dependent features specify NRP1-reactive agent options, separation modalities, and an MLC2v+ outcome.
Timed Wnt/β-catenin signaling modulation to obtain a cardiac progenitor-containing culture
Subjecting human pluripotent stem cells to Wnt/β-catenin signaling activation on day 0 of culture, followed by inhibition of Wnt/β-catenin signaling activation on day 3 to day 5 of culture to obtain a culture of human cells containing cardiac progenitor cells.
NRP1-reactive agent contacting during day 5-7
Contacting the culture of human cells containing cardiac progenitor cells with one or more agents reactive with neuropilin-1 (NRP1), wherein the culture is contacted with the one or more agents reactive with NRP1 on day 5-7 of culture.
Separation of NRP1-reactive positive cells
Separating NRP1 reactive positive cells from non-reactive cells to thereby isolate NRP1+ human cardiac ventricular progenitor cells.
Anti-NRP1 antibody as the NRP1-reactive agent
The method further includes contacting with an anti-NRP1 antibody as the agent reactive with NRP1.
Soluble NRP1 ligand as the NRP1-reactive agent
The method further includes that the agent reactive with NRP1 includes a soluble NRP1 ligand.
VEGF-A or Sema3A soluble NRP1 ligands
The NRP1 ligand is VEGF-A or Sema3A.
FACS or MACS separation of NRP1 reactive positive cells
Separating NRP1 reactive positive cells from non-reactive cells using fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS).
MLC2v+ outcome following culturing and differentiating
The method further includes culturing and differentiating NRP1+ human cardiac ventricular progenitor cells so that the resulting cells are MLC2v positive.
Overall, the claim set covers isolating NRP1+ human cardiac ventricular progenitor cells by generating a cardiac progenitor-containing culture via timed Wnt/β-catenin activation and inhibition, contacting the culture with NRP1-reactive agents during day 5-7, and separating NRP1 reactive positive cells. Dependent claims further specify NRP1-reactive agent types, separation via FACS or MACS, and an MLC2v+ differentiation or characterization outcome.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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