CN-122012383-A - Preparation method and application of iPSC directional differentiated myocardial cell mitochondria

Abstract

The invention discloses a preparation method and application of IPSC directional differentiation cardiomyocyte mitochondria, relating to the technical field of mitochondria, mitochondria were isolated from human Induced Pluripotent Stem Cells (IPSCs) by directed differentiation into cardiomyocytes (IPSCs-CMs). The preparation method comprises the steps of inducing and differentiating the iPSC into iPSC-CMs by utilizing a Wnt signal path regulation differentiation system, purifying by a metabolic selection method, and separating mitochondria by a homogenization method and a differential centrifugation method. The mitochondria prepared by the method can be effectively ingested by tubular epithelial cells, and can obviously relieve oxidative damage and/or renal ischemia reperfusion injury of the tubular epithelial cells through mechanisms of inhibiting Reactive Oxygen Species (ROS) accumulation, improving ATP synthesis capacity, recovering mitochondrial membrane potential, inhibiting apoptosis and the like.

Inventors

• SHANG WENJUN
• QI YUANBO
• GAO ZHEN
• WANG ZHIGANG

Assignees

• 郑州大学第一附属医院

Dates

Publication Date

20260512

Application Date

20260313

Claims (8)

1. The preparation method of the iPSC directional differentiated cardiomyocyte mitochondria is characterized by comprising the following steps: a. directionally differentiating human Induced Pluripotent Stem Cells (iPSCs) into myocardial cells (iPSCs-CMs); b. mitochondria were isolated and extracted from the iPSC-CMs.
2. 2. The method for preparing iPSC-directed differentiated cardiomyocyte mitochondria according to claim 1, wherein the step a comprises: a1. inoculating the human iPSC to a culture dish coated by matrigel; a2. When the cell density reaches 85% -95%, a Wnt signal channel is adopted to regulate and control a differentiation system for induction; a3. Obtaining autonomously pulsating myocardial cells after differentiation for 8-12 days; a4. purifying by metabolic selection method to obtain iPSC-CMs with purity not less than 85%.
3. 3. The method for preparing iPSC-directed differentiated cardiomyocyte mitochondria according to claim 1, wherein the step b comprises: b1. digestion and collection of iPSC-CMs, washing with pre-chilled phosphate buffer; b2. Adding mitochondrial separation buffer solution, adding steel balls with the diameter of 2mm, and homogenizing in a tissue grinder by 30Hz oscillation for 30s; b3.1000g for 5min, removing cell nucleus and fragments, and collecting supernatant; b4. centrifuging the supernatant in the range of 3500g to 12000g for 10min, collecting the precipitate as mitochondrial component; b5. The mitochondrial pellet was resuspended with mitochondrial storage.
4. 4. The method of claim 1, further comprising quality control of the resulting mitochondria, the quality control comprising one or more of: a. observing the integrity of the mitochondrial double-layer membrane by a transmission electron microscope; mito Tracker staining confirmed that mitochondrial membrane potential was intact; c. Cytotoxicity test.
5. 5. The method of claim 4, wherein the concentration of the mitochondrial preparation is 5 x 10 5 /mL.
6. 6. Use of mitochondria prepared according to the method of claim 1 for the preparation of a medicament for reducing oxidative damage to tubular epithelial cells and/or ischemia reperfusion injury of the kidney.
7. 7. The use of claim 6, wherein the mitochondria are delivered to the renal cortical region of the recipient by subrenal capsule injection.
8. 8. A pharmaceutical composition for treating oxidative damage and/or ischemia reperfusion injury of tubular epithelial cells, comprising the mitochondrial preparation of claim 5 and a pharmaceutically acceptable carrier.

Description

Preparation method and application of iPSC directional differentiated myocardial cell mitochondria Technical Field The invention relates to the technical field of mitochondria, in particular to a preparation method and application of IPSC directional differentiation cardiomyocyte mitochondria. Background Renal transplantation is the most effective treatment modality for end-stage renal disease (end-STAGE RENAL DISEASE, ESRD) patients, which significantly prolongs patient survival and improves quality of life. However, the inevitable ischemia reperfusion injury (ischemia-reperfusion injury, IRI) during transplantation severely affects early functional recovery and long-term survival of transplanted kidneys, and is a significant cause of delayed functional recovery (DELAYED GRAFT function, DGF) and chronic transplanted kidney dysfunction. There is currently no FDA approved drug specifically for the treatment of renal IRI. Kidneys belong to a hypermetabolic organ, in which the proximal Tubular Epithelial Cells (TEC) are most abundant in their mitochondrial content, and their energy is mainly derived from the Fatty Acid Oxidation (FAO) and oxidative phosphorylation (OXPHOS) pathways. During the ischemic phase, the interruption of oxygen supply resulted in a blocked electron transfer chain function, reduced ATP production, inactivation of Na +/K+ pumps, overload of intracellular Ca 2+, and abnormal opening of mitochondrial permeability transition pore (mPTP). During reperfusion, electron leakage is aggravated, active oxygen is produced in large amounts, mitochondrial membrane potential is lost, cytochrome c is released, and apoptosis and inflammatory cascade are activated. Numerous studies have shown that mitochondrial dysfunction is a critical node in the development and progression of IRI. Kidney IRI, which is a pathological process resulting from recovery of perfusion after interruption of tissue blood flow, can be used to simulate the pathological process of the cell layer in IRI by oxidative damage to TEC and hypoxia-reoxygenation damage in vitro experiments. In recent years, mitochondrial transplantation (mitochondrial transplantation) has received widespread attention as a novel organelle therapeutic strategy. This technique supplements mitochondrial numbers and restores metabolic function by isolating functionally intact mitochondria from healthy tissue and delivering to damaged tissue. Previous researches show that mitochondria can transfer among cells, mitochondrial transplantation can improve functional recovery in a myocardial IRI model, mitochondrial supplementation can relieve various metabolic diseases, and mitochondrial transplantation shows a certain protection effect in an in-vitro renal tubular injury model. However, the existing research is mainly focused on the current situations of skeletal muscle or autologous tissue source mitochondria, insufficient donor source stability, difficult standardization of preparation, lower purity and the like, and the application in kidney IRI is still in an exploration stage. Cardiomyocytes have a high mitochondrial content, a strong oxidative phosphorylation capacity, an active energy metabolism, and human induced pluripotent stem cells (Induced pluripotent STEM CELLS, iPSCs) refer to reprogramming terminally differentiated somatic cells into pluripotent stem cells by introducing specific transcription factors. Unlike classical embryonic stem cell technology and somatic cell nuclear transfer technology, iPSCs technology does not use embryonic or egg cells and therefore has no ethical problems. In addition, the proprietary stem cells can be prepared from the somatic cells of the patient by utilizing the iPSCs technology, so that the possibility of immune rejection reaction is greatly reduced. Under certain conditions, iPSCs can differentiate into cardiomyocytes in a targeted manner. If the iPSC induced differentiated myocardial cells can be used as a donor source, and a standardized separation and quality control system is combined, the mitochondrial preparation with high activity and large-scale preparation can be hopefully obtained, so that a new technical path is provided for IRI treatment. At present, no report has been made on the use of iPSC directional differentiated cardiomyocytes as a standardized mitochondrial donor source, the systematic verification of the effect of the source mitochondria in TEC oxidative damage and ischemia reperfusion models, and the construction of a mitochondrial preparation system with definite quality control indexes. Therefore, it is necessary to provide a method for preparing mitochondria of IPSC-directed differentiated cardiomyocytes and application thereof to solve the above problems. Disclosure of Invention The invention aims to provide a method for separating high-activity mitochondria from human Induced Pluripotent Stem Cell (iPSC) directional differentiated cardiomyocytes (iPSC-CMs), and provides application of th