Cell cycle block improves efficiency in generating induced pluripotent stem cells

A technology of pluripotent stem cells and cell cycle, which is applied in the field of cell cycle arrest to improve the efficiency of producing induced pluripotent stem cells, and can solve the problems of low efficiency

Inactive Publication Date: 2018-07-31
ORIG3N INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method used to generate iPSCs is well defined; however, it is less efficient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0049] Example 1. Harvesting of peripheral blood mononuclear cells.

[0050] With 3mL LeucoSep TM Separation medium (Greiner Bio One) was filled with 12 mL of LeucoSep TM test tube. The tubes were centrifuged at 1000 rcf for 30 seconds at room temperature to keep the separation medium in the tubes below the porous barrier.

[0051] Add 4 mL of phosphate-buffered saline (PBS; without calcium and magnesium) to a 15 mL conical test tube. Human blood in a 4 mL vacutainer was inverted 10 times to mix the blood. The blood is then added to the conical tube containing PBS, and the blood and PBS are mixed. Then pour the blood and PBS mixture into the LeucoSep TM test tube.

[0052] LeucoSep TM Tubes were centrifuged at 1250 rcf in a Labnet centrifuge (or 2100 rpm in a Beckman swinging bucket centrifuge) for 30 minutes at room temperature. Collect the cell-rich fraction, which contains lymphocytes and peripheral blood mononuclear cells, by decanting the plasma supernatant above ...

example 2

[0054] Example 2. Transduction of peripheral blood mononuclear cells.

[0055] A 0.5 mL aliquot of peripheral blood mononuclear cells was washed with 0.5 mL of expansion medium and placed in a 15 mL Erlenmeyer vial. Cells were pelleted at 250 rcf for 7 minutes, and the supernatant was decanted, leaving approximately 100 μL of medium in the tube.

[0056] Prepare transduction medium containing 0.4 mL of StemPro-34Lance Media; 5 μL of hKOS; 5 μL of hc-Myc; 3 μL of h-Klf4; 2 μL of Polybrene in water (1 mg / mL dilution); Reagent (10mg / mL).

[0057] Frozen CytoTune virus vials were placed in a 37°C bath for 8 seconds, causing the reagents to melt, and then placed in a 4°C cold block. Mix virus into PBMC expansion medium.

[0058] Next, transduction medium was placed in a 15 mL Erlenmeyer vial to resuspend the cell pellet. Place the transduction medium and cells in one well of a 24-well plate and store in 5% CO 2 Incubate overnight at 37°C in a humid atmosphere.

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PUM

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Abstract

Disclosed are methods for generating an induced pluripotent stem cell (iPSC) by arresting the cell cycle of a cell, and then transforming the cell to the iPSC.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to US Provisional Application No. 62 / 249,520, filed November 2, 2015, which is hereby incorporated by reference in its entirety. Background technique [0003] Stem cells are rare and difficult to isolate in appreciable quantities from adult tissues. In 2006, a research team led by Shinye Yamanaka published an article describing how differentiated adult cells can be reprogrammed to exhibit many stem cell properties, including pluripotency (Takahashi, K. & Yamanaka, Stem Cells (S.CELL) 126:663-676 (2006)). These "induced pluripotent stem cells" (iPSCs) can essentially replicate indefinitely, and they can differentiate into any cell type derived from the three embryonic germ layers to potentially form any cell in the human body. Thus, the development of iPSC methods has opened new avenues for personalized and regenerative medicine. The method used to generate iPSCs is well...

Claims

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Application Information

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IPC IPC(8): A61K35/545A61K35/12A61K35/35C12N5/071
CPCA61K35/545A61K35/17C12N5/0696C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2506/11C12N2510/00
Inventor R·Y·史密斯M·A·格利克斯曼N·F·扎伊迪
Owner ORIG3N INC
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