Induced pacemaker and Purkinje cells from adult stem cells
Inventors
Alt, Eckhard • KARIMI, Tahereh
Assignees
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Publication Number
US-9913865-B2
Publication Date
2018-03-13
Expiration Date
Abstract
Adult stem cells are reprogrammed to form pacemaker cells and Purkinje cells through the sequential activation of SHOX2>TBX5>HCN2. These Purkinje cells spontaneously surround and connect with the larger pacemaker cells, thus forming an induced sinoatrial body that produces funny current and can make cardiovascular tissues beat in a manner similar to a natural sinoatrial node.
Core Innovation
The invention reprograms adult adipose tissue-derived stem cells into induced non-contractile cardiomyocyte cells by inducing specific differentiation toward cardiac pacemaker cells and Purkinje cells. The method includes sequentially expressing SHOX2>TBX5>HCN2 in a population of adult adipose tissue-derived stem cells and growing the cells until cardiac pacemaker cells and Purkinje cells form. The induced differentiated cells are described as forming associated pacemaker/Purkinje networks and exhibiting spontaneous electrophysiology characterized by “funny current” (If).
The invention further supports pacemaker versus Purkinje populations within the induced cells by using differentiation outcomes associated with marker expression and electrophysiology. Marker expression including CX30.2 and HCN4 is described in connection with the induced pacemaker/Purkinje network behavior. The formation of an induced sinoatrial body / sinoatrial node (SAN) is described as a growth outcome. The described gene expression approach optionally includes additional transcription factors TBX3 and TBX18 alongside SHOX2>TBX5>HCN2.
Delivery and induction strategies are described as sequential gene activation implemented via gene delivery formats that include lentiviral vectors under sequential doxycycline control, and an alternative mRNA-based transfection approach. The invention also describes defining pacemaker versus Purkinje populations by size sorting, using explicit size constraints for separating induced populations. Potential therapeutic use is discussed in the context of arrhythmias, including sick sinus syndrome and tachycardic arrhythmias.
Claims Coverage
The independent claim is directed to a method that induces adult adipose tissue-derived stem cells to differentiate into induced non-contractile cardiomyocyte cells by sequential expression of SHOX2>TBX5>HCN2 followed by growth until cardiac pacemaker cells and Purkinje cells form. Additional inventive features are captured in dependent claim refinements related to extended sequential gene sets, delivery format, optional induced sinoatrial body formation, and size-based definition of pacemaker versus Purkinje populations.
Sequential gene expression of SHOX2>TBX5>HCN2 to induce differentiation
Sequentially expressing SHOX2>TBX5>HCN2 in a population of adult adipose tissue-derived stem cells to induce specific differentiation of the cells.
Growing until cardiac pacemaker cells and Purkinje cells form
Growing the cells until cardiac pacemaker cells and Purkinje cells form.
Extended sequential transcription factor set including TBX3 and TBX18
Sequentially expressing a gene set that extends beyond SHOX2>TBX5>HCN2 to include additional transcription factors TBX3 and TBX18 (in combination with SHOX2, TBX5, and HCN2 as described in dependent coverage).
Lentiviral expression vectors for sequential multi-gene delivery
Inducing the sequential expression using one or more lentiviral expression vectors encoding SHOX2, TBX3, TBX5, TBX18, and HCN2.
mRNA-based inducing step encoding SHOX2, TBX3, TBX5, TBX18, and HCN2
Inducing differentiation using an inducing step that uses mRNA encoding SHOX2, TBX3, TBX5, TBX18, and HCN2.
Formation of an induced sinoatrial body
Growing pacemaker cells and Purkinje cells until an induced sinoatrial body is formed.
Size sorting to define pacemaker versus Purkinje populations
Separating by size such that cells below 20 µm are treated as Purkinje cells and cells above 50 µm are treated as pacemaker cells.
Across the claim set provided, the coverage centers on sequentially expressing SHOX2>TBX5>HCN2 (with dependent extensions that include TBX3 and TBX18) in adult adipose tissue-derived stem cells, followed by growth until pacemaker and Purkinje cells form. Dependent features further specify gene-delivery formats (lentiviral vectors or mRNA), an induced sinoatrial body formation endpoint, and size-sorting criteria for defining pacemaker versus Purkinje populations.
Stated Advantages
Induced formation of cardiac pacemaker cells and Purkinje cells from adult adipose tissue-derived stem cells.
Spontaneous electrophysiology characterized by “funny current” (If) in the induced pacemaker/Purkinje network.
Induced sinoatrial body / sinoatrial node (SAN) formation as an output of growth.
Marker expression including CX30.2 and HCN4 associated with the induced pacemaker/Purkinje network.
Ability to define pacemaker versus Purkinje populations by size sorting with explicit size thresholds.
Potential therapeutic use in arrhythmias including sick sinus syndrome and tachycardic arrhythmias.
Documented Applications
Therapeutic use in arrhythmias, including sick sinus syndrome and tachycardic arrhythmias.
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