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Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof

A technology of somatic cells and stem cells, applied in the field of biomedicine, can solve the problems of lack of donor tissue sources and loss of clinical effects

Inactive Publication Date: 2012-04-04
林雄斌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the serious shortage of donor tissue sources and the immune rejection after allogeneic pancreas or allogeneic / xenogeneic islet transplantation, the clinical effect was lost

Method used

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  • Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof
  • Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof
  • Method and kit allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Production of plant and protein-induced pancreatic stem cells using peripheral venous blood / umbilical cord blood as raw material cells

[0065] Sterile collection of peripheral venous blood and umbilical cord blood (donated by scientific research staff, Beijing Wuzhou Women's Hospital, etc.). Before collecting peripheral venous blood / umbilical cord blood, the consent of the blood donor or his immediate family members should be obtained, and the genetic and infectious disease history of the blood donor and his family, as well as all relevant virus test results in the hospital should be recorded.

[0066] Peripheral venous blood / cord blood was routinely anticoagulated and stored at 4°C. Send to the stem cell production center within 24 hours. After the center needs to do the corresponding virus detection again, it will enter the computer registration procedure of the center, and after recording the corresponding barcode number, the blood sample will enter th...

Embodiment 2

[0071] Example 2: Using peripheral venous blood / umbilical cord blood as raw material cells to produce autologous islets

[0072] The plant and protein-induced autologous pancreatic stem cells obtained in Example 1 were differentiated to produce autologous islets.

[0073] After culturing in cell culture medium C2 to form plant and protein-induced pancreatic stem cells, use C3 cell culture medium to continue culturing for 9-12 days to form islets. The mononuclear cells of 200ml peripheral venous blood / 80ml umbilical cord blood can reach 0.6-1×10 in the 9th-12th day after culturing in cell culture medium C3, and the yield of plant and protein-induced pancreatic cells 11 .

[0074] The resulting autologous islets were tested. Cellular immunofluorescence technique was used to detect the expression of insulin protein (for details, refer to editor-in-chief of Shen Guan et al., "Modern Immunology Experimental Technology", 2nd edition, Hubei Science and Technology Press, 1998), an...

Embodiment 3

[0077] In this example, the safety research on the transplantation of plant and protein-induced autologous pancreatic stem cells prepared in Example 1 into nude mice is as follows.

[0078] In two groups of nude mice transplantation experiments, nude mice were subcutaneously transplanted and injected with 1×10 8 plant and protein-induced pancreatic stem cells or 1 x 10 8 Personal natural pancreatic stem cells (gifted by the Reproductive Center of Hainan Medical College) were used to observe whether there is tumor formation at the inoculation site. Continuous observation for 90 days.

[0079] The results showed that none of the 10 nude mice in the plant and protein-induced pancreatic stem cell test group developed tumors, which was relatively safe; while all the 10 nude mice in the human natural embryonic stem cell test group developed tumors.

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Abstract

The invention provides a method allowing reverse differentiation of human somatic cells for generation of autologous pancreatic stem cells and autologous pancreas islet. The method is as follows: culture solutions containing extractives of different plants and different protein components are used to culture human somatic cells step by step, which enables the somatic cells to generate autologous pancreatic stem cells through reverse differentiation, and autologous pancreas islet is generated through further culture. The method provided in the invention enables the first generation of autologous pancreatic stem cells and autologous pancreas islet at a magnitude order of tens of billions to be generated within two to three weeks. The plant and protein-induced human autologous pancreatic stem cells have similar cell specific phenotypes and a plurality of cell differentiation capability as those of human natural pancreatic stem cells; the autologous pancreatic stem cells and autologous pancreas islet cells have immense potential both in the application field of treatment of pancreatic diseases like insulin deficiency type diabetes and in the engineering field of autologous pancreatic tissue; the invention is also applicable to establishment of novel autologous pancreatic stem cell libraries and long-term permanent preservation of autologous pancreatic stem cells.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular, the invention relates to a cell culture method and application thereof for reversely differentiating human somatic cells to generate autologous pancreatic stem cells and autologous islets. Background technique [0002] During the development of the pancreas during the embryonic period, pluripotent stem cells gradually differentiate into several endocrine cells, the earliest appearing cells that produce glucagon and insulin, that is, islets of Langerhans. By 2-3 weeks after birth, the outline of the islets has become clear and mature; at this time, the activity of pancreatic pluripotent stem cells can hardly be seen in the pancreas, and the activity of producing new islets has stopped; not only that, due to the insulin production in the pancreas The regenerative ability of islet cells is very weak, causing various etiologies of insulin-dependent diabetes mellitus (IDDM), resulti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/078C12N5/073C12N5/077C12N5/074A61K35/39A61P5/00A61P25/00A61P1/16A61P13/12A61P1/18A61P3/10
CPCC12N5/0676A61P1/16A61P1/18A61P3/10A61P5/00A61P13/12A61P25/00
Inventor 林雄斌
Owner 林雄斌
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