Search

EP-4741496-A1 - METHOD FOR REPROGRAMMING HUMAN SOMATIC CELLS INTO INDUCED PLURIPOTENT STEM CELLS

EP4741496A1EP 4741496 A1EP4741496 A1EP 4741496A1EP-4741496-A1

Abstract

A simpler and more efficient method for reprogramming human somatic cells into induced pluripotent stem cells. According to the method, three transcription factors of OCT4, SOX2, and LIN28 are simultaneously transferred into somatic cells, and then reprogramming is performed under the co-induction of four small molecules of Peficitinib, VTP50469, SB203580, and FPFT-2216, wherein the peripheral blood reprogramming efficiency thereof can reach 0.02%, the induction period is only 14-16 days, and the system is stable.

Inventors

  • GUO, Ying
  • YANG, Yixing

Assignees

  • Exoneuglia Biotechnology (Suzhou) Co., Ltd.

Dates

Publication Date
20260513
Application Date
20240819

Claims (9)

  1. A method of reprogramming human somatic cells into induced pluripotent stem cells, characterized by comprising the following steps: introducing only three reprogramming induction factors, OCT4, SOX2, and LIN28, into the somatic cells, and culturing in the presence of chemical inducers to obtain induced pluripotent stem cells; wherein the chemical inducers consist of Peficitinib, VTP50469, SB203580, and FPFT-2216; and wherein a concentration of Peficitinib is 0.5 µM to 1.5 µM, a concentration of VTP50469 is 0.1 µM to 1 µM, a concentration of SB203580 is 1 µM to 3 µM, and a concentration of FPFT-2216 is 1 µM to 5 µM.
  2. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 1, characterized in that the three reprogramming induction factors OCT4, SOX2, and LIN28 are introduced into the somatic cells in the form of nucleic acids thereof or protein products thereof.
  3. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 1, characterized in that the three reprogramming induction factors OCT4, SOX2, and LIN28 are introduced into the somatic cells in the form of DNA or mRNA thereof.
  4. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 1, characterized in that the form for introducing the three reprogramming induction factors OCT4, SOX2, and LIN28 into somatic cells includes, but is not limited to, a form of Sendai virus transfection system, an mRNA transfection system, or an episomal plasmid transfection.
  5. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 1, characterized in that the somatic cells are hematopoietic precursor cells derived from peripheral blood.
  6. The method of reprogramming human somatic cells into induced pluripotent stem cells according to any one of claims 1 to 5, characterized by comprising the following steps: introducing the three transcription factors OCT4, SOX2, and LIN28 into somatic cells; inducing by using an induction medium containing the chemical inducers of claim 1; and finally culturing by using a pluripotent stem cell medium until the induced pluripotent stem cell clones are fully mature, followed by randomly picking clones for expansion; wherein, the induction medium is a pluripotent stem cell medium supplemented with 2 µM to 4 µM chir99021, 80 ng/ml to 120 ng/ml FGF2, 200 µM to 300 µM NaB, 1 µM to 3 µM SB203580, 0.5 µM to 1.5 µM peficitinib, 0.05 µM to 0.2 µM VTP50469, 1 µM to 5 µM FPFT-2216, 1% to 3% (v/v) B27, and 1% (v/v) N2; wherein the pluripotent stem cell medium is mTeSR1 medium.
  7. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 6, characterized by comprising introducing the three transcription factors OCT4, SOX2, and LIN28 into somatic cells via electroporation of episomal plasmid, wherein the episomal plasmid is a modified pCEP4 vector carrying the three transcription factors, and wherein the two transcription factors OCT4 and SOX2 are constructed on the same pCEP4 vector.
  8. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 7, characterized by comprising: immediately adding peripheral blood expansion medium after electroporation, seeding the cells in a plate coated with basement membrane matrix, and culturing for 46 h to 50 h; adding an induction medium and culturing for another 46 h to 50 h; subsequently performing partial medium changes with fresh induction medium and continuing induction for 46 h to 50 h, repeated 1 to 3 times; completely removing all the old medium and adding pluripotent stem cell medium for culture; replacing the pluripotent stem cell medium daily until the induced pluripotent stem cell clones are fully mature; and picking clones for expansion at any time.
  9. The method of reprogramming human somatic cells into induced pluripotent stem cells according to claim 8, characterized in that the peripheral blood expansion medium is a serum-free medium supplemented with 80 ng/mL to 120 ng/mL SCF, 80 ng/mL to 120 ng/mL FLT-3, 10 ng/mL to 30 ng/mL IL-3, 10 ng/mL to 30 ng/mL IL-6, and 80 ng/mL to 120 ng/mL TPO.

Description

TECHNICAL FIELD The present invention relates to the technical field of pluripotent stem cell reprogramming, specifically to a simpler and more efficient method for reprogramming human somatic cells into induced pluripotent stem cells. BACKGROUND ART Since Shinya Yamanaka's research group first induced mouse fibroblasts into pluripotent stem cells in 2006, the technology of pluripotent stem cell reprogramming has undergone rapid development. The reprogramming of human somatic cells has also progressively been achieved. For example, Junying Yu's research group has reprogrammed peripheral blood cells into induced pluripotent stem cells by introducing six transcription factors: OCT4, SOX2, LIN28, NANOG, C-MYC and KLF4. The research groups of Miguel Esteban and Ruiqing Pei successfully reprogrammed renal epithelial cells into induced pluripotent stem cells by introducing three transcription factors (OCT4, SOX2, and KLF4) and microRNA302/367 into urinary cells. In 2022, Hongkui Deng's research group successfully reprogrammed human-derived fibroblasts into pluripotent stem cells using a complete small-molecule reprogramming approach; however, the induction process was extremely time-consuming. The process of reprogramming of somatic cells into induced pluripotent stem cells involves drastic transitions in both cell fate and characteristic genes expression. Currently, scientists have a general understanding of the genes that are completely suppressed and that are completely activated following somatic-to-stem cell conversion. By introducing small molecules that promote pathways for target gene activation and inhibit pathways for silencing undesirable genes, it is possible to achieve an efficient and stable reprogramming scheme with minimal transcription factors. However, research progress in reprogramming with minimal transcription factors currently remains slow. Of the few schemes that have been reported, issues such as poor reproducibility and inconsistent efficiency still exist. For example, Chinese invention patent (Publication No. CN114645023B), titled "System and method of reprogramming peripheral blood mononuclear cells into induced pluripotent stem cells", has a reprogramming efficiency of only about 0.006%. Furthermore, the number of clones varies significantly in multiple replicate experiments, with efficiency fluctuating between 0.002% and 0.006%, indicating substantial instability. Therefore, to gain deeper insights into the roles of various transcription factors in reprogramming somatic cells into embryonic stem cells, and to improve reprogramming schemes for somatic cell-to-embryonic stem cell conversion, it is necessary to develop a highly efficient and reproducible reprogramming scheme that is particularly suitable for difficult-to-transfect cell types, such as peripheral blood cells. SUMMARY Based on this, one objective of the present invention is to provide a method of reprogramming human somatic cells into induced pluripotent stem cells, comprising the following steps: introducing only three reprogramming induction factors OCT4, SOX2, and LIN28 into somatic cells, and culturing the somatic cells in the presence of chemical inducers to obtain induced pluripotent stem cells;wherein the chemical inducers consist of Peficitinib, VTP50469, SB203580, and FPFT-2216; and wherein a concentration of Peficitinib is 0.5 µM to 1.5 µM, a concentration of VTP50469 is 0.1 µM to 1 µM, a concentration of SB203580 is 1 µM to 3 µM, and a concentration of FPFT-2216 is 1 µM to 5 µM. In a preferred embodiment, the three reprogramming induction factors OCT4, SOX2, and LIN28 are introduced into the somatic cells in the form of nucleic acids thereof or protein products thereof. In a preferred embodiment, the three reprogramming induction factors OCT4, SOX2, and LIN28 are introduced into the somatic cells in the form of DNA or mRNA thereof. In a preferred embodiment, the form of introducing the three reprogramming induction factors OCT4, SOX2, and LIN28 into somatic cells includes, but is not limited to, a form of Sendai virus transfection system, an mRNA transfection system, or an episomal plasmid transfection. In a preferred embodiment, the somatic cells are hematopoietic precursor cells derived from peripheral blood. In a preferred embodiment, the method of reprogramming human somatic cells into induced pluripotent stem cells comprises the following steps: introducing the three transcription factors OCT4, SOX2, and LIN28 into somatic cells; inducing the somatic cells by using an induction medium containing the chemical inducers of claim 1; and finally culturing the somatic cells by using a pluripotent stem cell medium until the induced pluripotent stem cell clones are fully mature, followed by randomly picking clones for expansion;wherein, the induction medium is a pluripotent stem cell medium supplemented with 2 µM to 4 µM chir99021, 80 ng/ml to 120 ng/ml FGF2, 200 µM to 300 µM NaB, 1 µM to 3 µM SB203580, 0.5 µM to 1.