Use of LIFR or FGFR3 as a cell surface marker for isolating human cardiac ventricular progenitor cells
Inventors
Chien, Kenneth R. • LIAN, Xiaojun Lance
Assignees
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Publication Number
US-10596200-B2
Publication Date
2020-03-24
Expiration Date
Abstract
The present invention provides LIFR and FGFR3 as cell surface markers for isolating human cardiomyogenic ventricular progenitor cells, in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Thus, the invention provides human ventricular progenitor (HVP) cells. The invention provides in vitro methods of the separation of Islet 1+ LIFR+ ventricular progenitor cells and/or Islet 1+/FGFR3+ ventricular progenitor cells and/or Islet 1+/LIFR+/FGFR3+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated LIFR+ and/or FGFR3+ ventricular progenitor cells are also provided. Methods of in vivo use of LIFR+ and/or FGFR3+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the LIFR+ and/or FGFR3+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.
Core Innovation
The patent describes discovering Leukemia Inhibitory Factor Receptor (LIFR) and Fibroblast Growth Factor Receptor 3 (FGFR3) as cell-surface markers for isolating human cardiac ventricular progenitor (HVP) cells biased toward the ventricular lineage. The invention uses Islet 1 (ISL1) in combination with LIFR/FGFR3 to define and enrich populations of ventricular progenitors.
Using the defined LIFR and FGFR3 marker combination, the patent describes scalable in vitro separation and expansion to clonal populations, including clonal populations of 1×10^9+ cells. The isolated LIFR+ and/or FGFR3+ progenitors are presented as suitable for both in vitro and in vivo downstream uses aimed at cardiac repair and function.
The patent further documents in vivo applications including organ-on-organ tissue engineering, described as 3D vascularized ventricular muscle on kidney capsule or epicardium, using purified LIFR+/FGFR3+ progenitors. Additional described uses include cardiac toxicity screening using purified LIFR+/FGFR3+ progenitors and generating HVPs via temporal Wnt/β-catenin activation/inhibition, with differentiation readouts such as MLC2v.
Claims Coverage
The independent claim is directed to isolating human cardiac ventricular progenitor cells using a temporal Wnt/β-catenin activation-to-inhibition differentiation sequence followed by marker-reactive contacting and separation for LIFR+ and/or FGFR3+ cells. Dependent claims refine the contacting and separation by adding Islet 1 and by specifying particular LIFR/FGFR3-reactive agents and cell-sorting modalities.
Temporal Wnt/β-catenin activation and inhibition to generate CPCs
Activating Wnt/β-catenin signaling at day 0, inhibiting Wnt/β-catenin signaling at day 3-5 to generate human cardiac progenitor cells (CPCs), and then contacting the human CPCs at day 5-7 with one or more agents reactive with LIFR and/or FGFR3.
LIFR- and/or FGFR3-reactive contacting and isolation from negative cells
Contacting the human CPCs with one or more agents reactive with Leukemia Inhibitory Factor Receptor (LIFR) and/or Fibroblast Growth Factor Receptor 3 (FGFR3), separating LIFR+ cells and/or FGFR3+ cells from negative cells, and isolating the LIFR+ cells and/or FGFR3+ cells as LIFR+ and/or FGFR3+ human cardiac ventricular progenitor cells.
Islet 1 combined with LIFR/FGFR3 isolation
Contacting the human CPCs with an agent reactive with Islet 1, separating LIFR+ and/or FGFR3+/Islet 1+ cells from negative cells, and isolating the LIFR+ and/or FGFR3+/Islet 1+ human cardiac ventricular progenitor cells.
Agent reactive with anti-FGFR3 antibody
At step 4, contacting the human CPCs with an anti-FGFR3 antibody.
Agent reactive with anti-LIFR antibody
At step 4, contacting the human CPCs with an anti-LIFR antibody.
Ordering constraint for Islet 1-reactive then LIFR/FGFR3-reactive contacting
Contacting the human CPCs with the agent reactive with Islet 1 before contacting them with one or more agents reactive with LIFR and/or FGFR3.
Separation by fluorescence activated or magnetic activated cell sorting
Separating the LIFR+ cells and/or FGFR3+ cells from negative cells by fluorescence activated cell sorting or magnetic activated cell sorting.
Across the independent and dependent claim set, the core approach is to generate human cardiac progenitor cells using temporal Wnt/β-catenin signaling, then enrich for human cardiac ventricular progenitor cells by contacting with LIFR- and/or FGFR3-reactive agents and isolating LIFR+ and/or FGFR3+ cells, with refinement including Islet 1 integration, specific LIFR/FGFR3-reactive agents, and FACS or MACS separation.
Stated Advantages
Enables scalable in vitro separation and expansion to clonal populations, including clonal populations of 1×10^9+ cells.
Supports in vivo organ-on-organ tissue engineering, described as 3D vascularized ventricular muscle on kidney capsule or epicardium.
Supports cardiac toxicity screening using purified LIFR+/FGFR3+ progenitors.
Supports generating HVPs and includes differentiation readouts such as MLC2v.
Supports improved cardiac repair/function (described in the provided content).
Documented Applications
Organ-on-organ tissue engineering using 3D vascularized ventricular muscle on kidney capsule or epicardium.
Cardiac toxicity screening using purified LIFR+/FGFR3+ progenitors.
Generating human cardiac ventricular progenitor cells (HVPs) via temporal Wnt/β-catenin activation/inhibition with differentiation readouts such as MLC2v.
In vivo cardiac repair/function (described in the provided content).
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