Use of neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells

A technology of cardiac progenitor cells and progenitor cells, applied in the field of the ventricular region of the heart, can solve problems such as isolating a large number of living cells, and the target is out of reach

Active Publication Date: 2020-05-08
PROCELLA THERAPEUTICS AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

To this end, various cardiac cell populations (atrial, ventricular, pacemaker) have been combined with artificial and decellularized matrices (Masumoto, H. et al. (2014) Sci. Rep. 4:5716; Ott, H.C. et al. (2008) Nat.Med.14:213-221; Schaaf, S. et al. (2011) PLoS One 6:e26397), Vascular cells and vessels (Tulloch, N.L. et al. (2011) Circ.Res.109: 47-59) and microRNA mixtures (Gama-Garvalho, M. et al. (2014) Cells 3:996-1026) have been investigated, but the target remains elusive
[0009] While the identification of Isl1 as a marker of cardiac progenitors is a major advance, because Isl1 is an intracellular protein, it is not an appropriate marker for isolating large numbers of living cells

Method used

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  • Use of neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells
  • Use of neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells
  • Use of neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells

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Experimental program
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Embodiment approach

[0204] In another embodiment, the method includes:

[0205] administering a test compound to a non-human animal, wherein the non-human animal comprises NRP1+ human ventricular progenitor cells transplanted into an organ of the non-human animal; and

[0206] The effect of test compounds on NRP1+ human ventricular progenitor cells in non-human animals is assessed.

[0207] In one embodiment, for example, by measuring the effect of a test compound on the viability of human ventricular tissue or NRP1+ human ventricular progenitor cells in a non-human animal (compared to the viability of the tissue or progenitor cells in the absence of the test compound), To assess the cardiotoxicity of test compounds. Cell viability can be assessed by standard methods known in the art.

[0208] In another embodiment, for example, by measuring the effect of the test compound on the differentiation of human ventricular tissue or NRP1+ progenitor cells in a non-human animal (compared to the differe...

Embodiment 1

[0215] Example 1: Generation of human Isl1+ cardiomyocyte-derived progenitor cells by modulating Wnt signaling in human pluripotent stem cells

[0216] Temporal modulation of canonical Wnt signaling has been shown to be sufficient to generate functional cardiomyocytes in high yield and purity from many hPSC lines (Lian, X. et al. (2012) Proc. Natl. Acad. Sci. USA 109 : E1848-1857; Lian, X. et al. (2013) Nat. Protoc. 8:162-175). In this approach, Wnt / β-catenin signaling is first activated in hPSCs, followed by an incubation period followed by inhibition of Wnt / β-catenin signaling. In the initially published protocol, by combining with the Gsk3 inhibitor CHIR99021 (GSK-3α, IC 50 =10nM; GSK-3, βIC50=6.7nM) to achieve Wnt / β-catenin signaling activation by incubation with Porcn inhibitor IWP2 (IC 50 = 27 nM) to achieve Wnt / β-catenin signaling inhibition. Because we used Gsk3 inhibitors and Wnt production inhibitors for cardiac differentiation, this protocol was called the GiWi p...

Embodiment 2

[0224] Example 2: Identification of Jagged 1 as a cell surface marker for cardiac progenitor cells

[0225]To characterize the transcriptional changes that occur during cardiac differentiation at the genome-scale level, RNA sequencing (RNA-seq) was performed at various time points after differentiation to establish the cardiac developmental transcriptional environment. We performed RNA-seq experiments on samples from days 0 to 7, as well as samples from days 19 and 35 (two independent biological replicates for each time point). Two batches of RNA-seq (100bp and 50bp read lengths) were performed using the illumine Hiseq 2000 platform. A total of 20 samples were examined. Mapped our reads into the reference human genome (hg19) using Bowtie and Tophat and calculated per-gene expression (genes were annotated according to Refseq) using the RPKM method (reads per kilobase transcript per million reads) . The differentiation of hPSCs into cardiomyocytes involves five major cell typ...

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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.

Description

[0001] related application [0002] This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 549,345, filed August 23, 2017. The content of this provisional application is incorporated herein by reference in its entirety. Background technique [0003] Heart failure, primarily caused by myocardial infarction, is the leading cause of death in adults and children worldwide and is growing exponentially worldwide (Bui, A.L. et al. (2011) Nat. Rev. Cardiol. 8:30-41). The disease is primarily driven by the loss of ventricular muscle that occurs during myocardial injury (Lin, Z. and Pu, W.T. (2014) Sci.Transl.Med.6:239rv1) and due to the negligible capacity of the adult heart to increase the regenerative response exacerbation (Bergmann, O. et al. (2009) Science 324:98-102; Senyo, S.E. et al. (2013) Nature 493:433-436). Although heart transplantation is curative, the apparently limited supply of human cardiac organ donors has resulted in a gener...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/077C12N5/0775C07K16/28
CPCC12N5/0657C12N2500/98C12N2501/415C12N2501/727C12N2506/02C12N2506/45C12N2533/90C12N15/00G01N33/5014G01N33/5044C07K16/2863A01K67/0275A01K2227/105A01K2267/0375A01K2267/0393C12N5/0605
Inventor K·R·钱J·克拉克C·Y·梁
Owner PROCELLA THERAPEUTICS AB
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