Human induced pluripotent stem cell as well as preparation method and application thereof

A technology of pluripotent stem cells and somatic cells, applied in the field of human induced pluripotent stem cells and its preparation, can solve the problem that animal models cannot completely simulate human disease states and therapeutic effects

Pending Publication Date: 2022-04-15
SHANXI MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Hemophilia is a monogenic genetic disease, and gene therapy is the only cure for it. Currently, the clinical trials of gene therapy are conducted on animals. Due to species differences, animal models cannot fully simulate the disease state and therapeutic effect of humans.

Method used

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  • Human induced pluripotent stem cell as well as preparation method and application thereof
  • Human induced pluripotent stem cell as well as preparation method and application thereof
  • Human induced pluripotent stem cell as well as preparation method and application thereof

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preparation example Construction

[0029] In some of these embodiments, the preparation method of the human induced pluripotent stem cell line SXMUi001-A-1 includes: the preparation method of the human induced pluripotent stem cell line SXMUi001-A-1 includes:

[0030] Introduce reprogramming factors into somatic cells of hemophilia A patients with c.2440C>T nonsense mutation in the F8 gene; the above-mentioned reprogramming factors include OCT4, SOX2, KLF4, MYC

[0031] Carry out cell reprogramming and obtain by screening.

[0032] In some embodiments, the above-mentioned somatic cells include but are not limited to urine, blood, adipose tissue, amniotic fluid, placental chorion, amniotic membrane, umbilical cord blood, periosteal cells or skin fibroblasts from various parts.

[0033] In some of these embodiments, the above-mentioned somatic cells are urine cells, which avoids the pain of sampling from the skin and the harm to the patient.

[0034] In some of these embodiments, the above-mentioned reprogrammin...

Embodiment 1

[0050] The inventor screened the gene mutation types of hemophilia patients in Shanxi area in the early stage. Among them, a nonsense mutation F8 c.2440C>T was detected in a hemophilia A patient in the Second Hospital of Shanxi Medical University, which was confirmed (http : / / www.factorix.org / ) This mutation is a nonsense mutation.

[0051] 1. Preparation of human induced pluripotent stem cells

[0052] (1) Collect urine cells:

[0053] With the consent of the patient, urine exfoliated cells were isolated from the urine samples of hemophilia patients, and the urine exfoliated cells were proliferated and cultured [Zhou, T., C. Benda, S. Dunzinger, Y. Huang, J. C. Ho, J. Yang, Y. Wang, Y. Zhang, Q. Zhuang, Y. Li, X. Bao, H.F. Tse, J. Grillari, R. Grillari-Voglauer, D. Pei, and M.A. Esteban. 2012.'Generation of human induced pluripotent stem cells from urine samples', Nat Protoc, 7:2080-9.].

[0054] Collect mid-section urine, centrifuge at 400g for 10 minutes at room temperat...

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Abstract

The invention relates to a human induced pluripotent stem cell strain SXMUi001-A-1 as well as a preparation method and application thereof, the human induced pluripotent stem cell strain SXMUi001-A-1 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No: 23095. The human induced pluripotent stem cell strain SXMUi001-A-1 is obtained by extracting urine cells from urine of a type A hemophilia patient carrying F8c.2440C > T (p.R814X) nonsense mutation and reprogramming, is similar to an embryonic stem cell in the aspects of cellular morphology, gene expression, proliferation and differentiation potential, and has the potential of differentiating to three germ layers. The human induced pluripotent stem cell strain SXMUi001-A-1 prepared by the invention has important value for molecular mechanism research of hemophilia nonsense mutation, detection of hemophilia, drug screening, gene therapy and other clinical application researches.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human induced pluripotent stem cell and its preparation method and application. Background technique [0002] Hemophilia is an X-chromosome-linked recessive hereditary hemorrhagic disease, the common features are active thromboplastin generation disorder, prolongation of coagulation time, lifelong tendency to bleeding after minor trauma, severe patients can also occur "spontaneous" without obvious trauma sex" bleeding. Including, hemophilia A (hemophilia A), that is, factor VIII procoagulant component (VIII: C) deficiency, also known as AGH deficiency; hemophilia B (hemophilia B), that is, factor IX deficiency, Also known as PTC deficiency, thromboplastin component deficiency; hemophilia C (hemophilia C), namely factor Ⅺ (FⅪ) deficiency, also known as PTA deficiency, thromboplastin precursor deficiency. [0003] Among them, hemophilia A is an X-chromosomal recessive genetic bleed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/071C12N15/85
CPCC12N5/0696C12N15/85C12N2501/603C12N2501/602C12N2501/604C12N2501/606C12N2506/25
Inventor 王刚杨林花孙雯雯姚明泽吴长新刘秀娥任娟张睿娟张夏林马艳春赵丽东
Owner SHANXI MEDICAL UNIV
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