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Method for orienting inducing and differentiating heart pacemaker cell by embryo source pluripotent stem cell

A technology of pluripotent stem cells and cardiac pacing, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., to achieve strong activity

Inactive Publication Date: 2011-08-03
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no report on the use of this cell to induce differentiation of cardiac pacemaker cells to construct a biological cardiac pacemaker

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Inducing canine embryo-derived pluripotent stem cells to prepare pacemaker cells

[0023] 1. Isolation, culture and identification of embryo-derived pluripotent stem cells

[0024] (1) Isolation and culture of embryonic-derived pluripotent stem cells

[0025] Canine whole bone marrow direct adherence method: extract 10ml of iliac bone marrow from adult hybrid dogs (Experimental Animal Center, Second Military Medical University, 15kg), centrifuge at 900×g for 10min, discard supernatant and fat; add red blood cell lysate TrisNH 1 Cl, remove red blood cells at 4°C for 5 min, add 1×10 5 / ml was inoculated in a culture dish coated with fibronectin (FN) (Sigma Company), and the embryo-derived pluripotent stem cell medium was 60% DMEM-LG (Gibco Company), 40% MCDB-201 added ITS (Sigma Company ), LA-BSA (BOVOGEN), 10mol / L dexamethasone (Sigma Company), 2% FCS (Gibco Company) and 10ng / ml PDGF-BB (Oncogene Company), 10ng / mlEGF (cytolab Company), 1000U / ml LIF (Chemico...

Embodiment 2

[0035] Example 2: Inducing rat embryo-derived pluripotent stem cells to prepare pacemaker cells

[0036] 1. Isolation, culture and identification of embryo-derived pluripotent stem cells

[0037] (1) Isolation and culture of rat embryo-derived pluripotent stem cells

[0038] Rat whole bone marrow direct adherence method: extract 10ml bone marrow from adult SD rats (Experimental Animal Center, Second Military Medical University, 250g), centrifuge at 900×g for 10min, discard supernatant and fat; add red blood cell lysate TrisNH 4 Cl, remove red blood cells at 4°C for 5 min, add 1×10 5 / ml was inoculated in a culture dish coated with fibronectin (FN) (Sigma Company), and the embryo-derived pluripotent stem cell medium was 60% DMEM-LG (Gibco Company), 40% MCDB-201 added ITS (Sigma Company ), LA-BSA (BOVOGEN), 10mol / L dexamethasone (Sigma Company), 2% FCS (Gibco Company) and 10ng / ml PDGF-BB (Oncogene Company), 10ng / ml EGF (cytolab Company), 1000U / ml LIF (Chemicon). After 24 ho...

Embodiment 3

[0049] Example 3: In vivo observation of pacing effect by transplantation of pacemaker cells prepared from rat embryo-derived pluripotent stem cells

[0050] The successfully induced pacemaker cells were injected into the ventricle wall of SD rats (Experimental Animal Center, Second Military Medical University, 250 g) using a micro-injection method, and cyclosporine A (Beijing Shuanglu Pharmaceutical Co., Ltd.) was applied 3 days before the injection. company, the dose is 0.25mg / day) and prednisolone (Shanghai General Pharmaceutical Co., Ltd., the dose is 0.1mg / day) to suppress immune rejection. After 2 weeks, the heart wall of the free graft area in the thoracic cavity was opened; the spontaneous beating of the free wall lasted for 10 minutes when placed in normal saline. 4% paraformaldehyde (Shanghai Chemical Reagent Co., Ltd., China Pharmaceutical) fixed the heart tissue in the transplanted area, made paraffin sections, and immunofluorescence detected the expression of Cx40...

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PUM

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Abstract

Cell transplantation for constructing biological heart pacemaker is used embryo source dry / ancestral cell and adult mesogalia dry cell as cell sources. The process is carried out by oriented differentiation inducing for heart pacemaker cell by embryo source multifunctional dry cell, bone marrow adhering to obtain primary culture cell, clone culturing to obtain purifying system by diluting, applying paracrine factor endothelin-1 or nervous adjusting protein-1, extracellular matrix laminated adherent protein and fiber connecting protein etc. substrate glue, trans-dyeing pacemaker gene HCN4, andin-vitro inducing embryo source multifunctional dry cell. It is various and non-immunogenicity. It can be used to treat sick sinus syndrome.

Description

Technical field: [0001] The invention relates to the technical field of induction and differentiation of stem cells, and relates to a method for inducing differentiation of embryonic-derived pluripotent stem cells into cardiac pacemaker cells and an application thereof. Background technique: [0002] The methods of constructing biological cardiac pacemakers can be divided into gene therapy and cell transplantation. The method based on gene therapy is to directly introduce specific genes into the heart to create or improve its automatic rhythm. However, although the transfection of pacing genes improves and produces the self-discipline of cardiomyocytes, it has been initially proved that a biological cardiac pacemaker can be constructed. , but still faces huge challenges, such as the lack of long-term stable expression of foreign genes in vivo and the difficulty of effective regulation; the inability to obtain efficient, safe, and well-directed vector systems, etc. Using cel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 张喜张传森
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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