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Efficient induced pluripotent stem cell reprogramming method for blood cells

A technology for pluripotent stem cells and blood cells, applied in the field of high-efficiency induced pluripotent stem cell reprogramming, can solve the problems of abnormal karyotype, low reprogramming efficiency, low efficiency, etc., and achieve the effect of less accumulation of gene mutations and convenient source of samples

Pending Publication Date: 2018-05-29
安徽中盛溯源生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] Since the virus / plasmid vectors used to transduce foreign genes into donor cells will affect them in the reprogramming process, there are gene rearrangements, karyotype abnormalities, and epigenetic abnormalities in the clones screened by this method. Abnormalities and other phenomena, even with high risk of cancer
In 2008, Okita et al. obtained mouse iPSCs through multiple conventional plasmid transfection methods, but the operation was cumbersome and the reprogramming efficiency was low
However, the initial OriP / EBNA1 episomal carrier reprogramming method is inefficient and requires trophoblast cells, which is not conducive to the large-scale preparation of hiPSCs and the preparation of clinical-grade hiPSCs

Method used

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  • Efficient induced pluripotent stem cell reprogramming method for blood cells
  • Efficient induced pluripotent stem cell reprogramming method for blood cells
  • Efficient induced pluripotent stem cell reprogramming method for blood cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Episomal vector construction

[0074] As shown in Figure 2, three kinds of episomal vectors were constructed in this embodiment, all of which amplified the ORF sequence in the potency determinant gene by polymerase chain reaction (PCR), and inserted the ORF sequence into the vector containing OriP / The mammalian expression vector pCEP4 of EBNA1 was constructed as an episomal vector, and all three episomal vectors contained at least one internal ribosome entry size (IRES), among which the first episomal vector was pEP4-E -O2S-E-N2K, which sequentially contains the first promoter, POU5F1, IRES2, SOX2, the second promoter, NANOG, IRES2 and KLF4, and the second vector is pEP4-E-O2S-E-T2K, which sequentially contains The third promoter, POU5F1, IRES2, SOX2, the fourth promoter, SV40LT, IRES2 and KLF4, the third vector is pCEP4-M-2L, which contains the fifth promoter, MYC, IRES2 and LIN28A in sequence; the above-mentioned first The second, third and fourth promoters are all ...

Embodiment 2

[0076] 1. Acquisition of red blood cell progenitor cells

[0077] Collect a blood sample of at least 10 μl, transfer it to a lymphocyte separation tube, centrifuge, take the mononuclear cell layer, wash it twice with DPBS centrifugation, take a sample and count it, and take 0.5×10 cells according to the counting result. 6 Cells / ml seeded in 96, 48, 24, 12 or 6-well plate, add erythrocyte progenitor cell expansion medium, place at 37°C, 5% CO 2 cultured in an incubator. The same volume of fresh expansion medium as the initial medium was added to each well on the 4th and 8th day of expansion respectively.

[0078] 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 of ferrous sulfate, Ferric nitrate 0.2 μmol, lipoic acid 1 μmol, hydrocortisone 1 μmol, stem cell factor 100 μg, e...

Embodiment 3

[0082] episomal vector-induced reprogramming

[0083] a, recovery, after the erythrocyte progenitor cells in Example 2 are overgrown, get 0.5~4×10 erythrocyte progenitor cells 6 , using the pEP4-E-O2S-E-N2K, pEP4-E-O2S-E-T2K and pCEP4-M-2L episomal vectors constructed in Example 1 to electrotransfect the above erythroid progenitor cells, and then inoculate them on induced pluripotent stem cells Culture medium and Matrigel or vitronectin or other cell matrix-coated six-well plate, the transfection content of each plasmid DNA is pEP4-E-O2S-E-N2K: pEP4-E-O2S-E-T2K : pCEP4-M-2L=1:1:1.

[0084] b. After 48 hours, half of the medium was replaced with fresh pluripotent stem cell induction medium, and the culture was continued until 10 days, and the medium was changed every other day, that is, reprogramming was carried out on the feeder-free system.

[0085] The pluripotent stem cell induction medium of this example has the following components: adding one or more of the following s...

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Abstract

The invention belongs to the field of cells and in particular relates to an efficient induced pluripotent stem cell reprogramming method for blood cells. The method comprises the following steps: S1,extracting monocyte from a blood specimen, and performing selective culture with an amplification culture medium so as to obtain erythrocyte progenitor cells; S2, introducing a free carrier with at least one potentiality determinant factor into the obtained erythrocyte progenitor cells; S3, culturing the erythrocyte progenitor cells with the free carrier by using a pluripotent stem cell inductionculture medium, and performing induction in a feeding layer free system so as to obtain reprogrammed intermediate state cells; S4, after induction is completed, replacing the pluripotent stem cell induction culture medium in the step S3 by a pluripotent stem cell culture medium to maintain culture, thereby obtaining cells that potentiality determinant factor expression is vanished and expression of endogenous pluripotent genes POU5F1, NANOG, TRA-1-60 and TRA-1-81 is activated, namely the induced pluripotent stem cells. The method has the beneficial effects that pluripotent stem cells without endogenous gene components can be efficiently induced, and the method is applicable to preclinical study and clinical application.

Description

technical field [0001] The invention belongs to the field of cells, and in particular relates to a high-efficiency induced pluripotent stem cell reprogramming method for blood cells. Background technique [0002] The inner cell mass of the blastocyst in the early stage of vertebrate embryonic development is pluripotent, and it can differentiate into all types of cells in the three germ layers of the body except the placenta. These terminally differentiated cells generally do not change their fate in vivo. Some studies have shown that re-differentiation of terminally differentiated cells into a pluripotent state can be achieved by means of nuclear transfer, cell fusion, and co-culture of pluripotent cell extracts, but these methods rely on scarce oocytes or existing pluripotent stem cells, so their applications are greatly limited. [0003] In 2006, the Yamanaka research group of Kyoto University in Japan used retroviral vectors to overexpress four exogenous transcription fa...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0696C12N2506/115C12N2501/60C12N2501/602C12N2501/605C12N2501/604C12N2501/608C12N2501/606Y02A50/30
Inventor 俞君英张健董成友孟晨吴文青张颖
Owner 安徽中盛溯源生物科技有限公司
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