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A method for reprogramming human blood cells into iPSCs via salamander Oct4

A blood cell and reprogramming technology, applied in the field of cells, can solve problems such as low efficiency, and achieve the effect of improving induction efficiency and convenient sample source.

Active Publication Date: 2019-08-27
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the OriP / EBNA1 episomal vector to reprogram human fibroblasts, we also found that human POU5F1 can be replaced by salamander Oct4, but the efficiency is also very low

Method used

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  • A method for reprogramming human blood cells into iPSCs via salamander Oct4
  • A method for reprogramming human blood cells into iPSCs via salamander Oct4
  • A method for reprogramming human blood cells into iPSCs via salamander Oct4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Obtaining the Coding Sequence of Transcription Factor Oct4 in Salamander

[0052] Amino acid sequence information encoded by the salamander Oct4 gene was obtained from http: / / www.ncbi.nlm.nih.gov / pubmed. The amino acid sequence encoded by the salamander Oct4 gene is SEQ ID NO: 1, see Sequence Table 1, and the corresponding coding sequence was synthesized by a commercial company.

Embodiment 2

[0054] Construction of Episomal Vector Expression System Containing Transcription Factor Encoded by OCT4

[0055]1. Construction of pEP4-E-OCT4 / AxOct4, pEP4-E-SCLM2L and pCEP4-ENET episomal vectors

[0056] Using traditional methods, the ORF sequence in the transcription factor gene was amplified by polymerase chain reaction and inserted into the mammalian expression vector pCEP4 containing OriP / EBNA1 to construct a reprogramming vector containing at least one internal ribosome entry site , wherein the pEP4-E-OCT4 / AxOct4 episomal vector contains the Amp resistance gene, the first promoter, OCT4 / AxOc4, the second promoter, and HcRed in sequence, and the pEP4-E-SCLM2L episomal vector contains the Amp resistance gene in sequence , the third promoter, SOX2, the fourth promoter, MYCL, IRES2, LIN28A, and wherein the pCEP4-ENET episomal vector contains the Amp resistance gene, the fifth promoter, NANOG, the sixth promoter, SV40LT in turn; wherein the first The first, third, fifth an...

Embodiment 3

[0058] Reprogramming of pluripotent stem cells derived from human blood cells

[0059] 1. Acquisition of red blood cell progenitor cells

[0060] A blood sample of at least 10 μl is collected, transferred to a lymphocyte separation tube, centrifuged, and a mononuclear cell layer is obtained, which includes neutrophils, eosinophils, basophils, lymphocytes, and erythroid progenitors etc., centrifuged and washed twice with DPBS, sampled and counted, and 3×10 6 Cells were inoculated into 6-well plates in 3 wells, added with 2 ml / well of erythrocyte progenitor cell expansion medium, and cultured in a 37° C., 5% CO2 incubator. Add 2ml of fresh expansion medium to each well on the 4th and 8th day of expansion respectively.

[0061] The specific formulation of the expansion medium in this example is: each liter of expansion medium contains 10ml of ITS additive, 10ml of GlutaMAX, 1ml of Lipid Concentrate, 250 μmol of L-ascorbic acid 2-phosphorylated hemimagnesium salt hydrate, 3 μmol...

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Abstract

The invention belongs to the field of cells and particularly relates to a method using salamander Oct4 to reprogram human blood cells into iPSC. The method includes the steps of S1, acquiring progenitor red blood cells; S2, importing a free vector containing a salamander Oct4 transcription factor and other transcription factors into the progenitor red blood cells obtained in S1; S3, subjecting theprogenitor red blood cells, containing the free vector, obtained in S2 to multipotential stem cell induction medium culture, and inducing into reprograming intermediate-state cells in a feed-layer-free system; S4, after complete induction, replacing the multipotential stem cell induction medium in S3 with multipotential stem cell culture medium to continue the culture so as to obtain the cells with disappeared salamander Oct4 transcription factor and other transcription factor expression and activated endogenous multipotential gene POU5F1, NANOG, TRA-1-60 and TRA-1-81 expression, wherein thecells are the iPSc. The method has the advantages that efficient induction can be performed to generate the hiPSC without exogenous gene components, and the method is applicable to preclinical study and clinical application.

Description

technical field [0001] The invention belongs to the field of cells, in particular to a method for reprogramming human blood cells into iPSCs through the newt Oct4. Background technique [0002] my country is a country with a large population in the world. Every year, organ defects, failures, and dysfunctions caused by trauma, disease, aging, and genetics also rank first in the world. Therefore, the research on stem cells and regenerative medicine has attracted the general attention of quite a few scientific research institutes and all walks of life. [0003] Cell transplantation therapy is an important direction of regenerative medicine research. Specific types of cell transplantation can be used to treat heart injury, nervous system degenerative disease, spinal cord injury, kidney failure, blood system disease and so on. However, cell transplantation therapy faces many difficult problems such as allogeneic rejection and limited cell sources. [0004] Stem cells are a type ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
Inventor 俞君英张健董成友张颖
Owner 安徽中盛溯源生物科技有限公司
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