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Method for directional differentiation of induced pluripotent stem cells into lymphoid tissue induced cells

A technology of pluripotent stem cells and lymphoid tissue, applied in the field of induced pluripotent stem cells to differentiate into lymphoid tissue induced cells, can solve the problems of low reproducibility, low differentiation efficiency, long cycle, etc., and achieve highly reproducible results

Active Publication Date: 2021-06-29
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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Problems solved by technology

Lymphoid tissue-induced cells (LTiCs) are a new type of natural lymphocytes that are essential for the development of peripheral lymphoid tissues in humans and mice. In the existing literature, studies involving lymphoid tissue-induced cells or natural immune lymphocytes often require the construction of transgenic mice , isolate lymphoid progenitor cells from a living body, and then induce cell differentiation in vitro by adding cytokines to induce different innate immune lymphocyte subsets and lymphoid tissues. low efficiency and low repeatability
In August 2006, Yamanaka's laboratory successfully induced mouse embryonic fibroblasts into "inducible pluripotent stem cells" similar to mouse embryonic stem cells by exogenously expressing four transcription factors, Oct4, Sox2, c-Myc, and Klf4. The results of this experiment indicate that differentiated cells can be induced by several transcription factors and recoded to a pluripotent state. At present, there is no relevant literature report on the induction of differentiated lymphoid tissue induced cells from mouse induced pluripotent stem cells through in vitro cloning culture

Method used

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  • Method for directional differentiation of induced pluripotent stem cells into lymphoid tissue induced cells
  • Method for directional differentiation of induced pluripotent stem cells into lymphoid tissue induced cells
  • Method for directional differentiation of induced pluripotent stem cells into lymphoid tissue induced cells

Examples

Experimental program
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Embodiment 1

[0037] Embodiment 1, mouse embryonic fibroblast (MEF) preparation induced pluripotent stem cell (iPS)

[0038] 1) Take C57 / BL6 mice aged 5-6 weeks, and put them together at 18:00 on the same day with a male-to-female ratio of 2:1. Check the mating situation of the female mice the next morning. The white vaginal plug in the vagina of the female mice is the sign of pregnancy. They were reared in separate cages and recorded as 0.5 days pregnant. Take the C57 / BL6 mouse embryos on the 13.5th day of pregnancy, remove the head / viscera and limbs of the embryos, add an appropriate amount of 0.25% trypsin-EDTA to digest and prepare mouse embryonic fibroblasts (MEFs), wash and centrifuge with 10 The complete culture medium of %FBS was seeded into T75 culture flasks and cultured in a 5% CO2 incubator at 37°C. After the cells grew to 85-90% confluence, they could be digested and passaged. The MEFs within the third generation were planted at a suitable density, and the lentivirus construct...

Embodiment 2

[0045] Example 2, Construction of Induced Pluripotent Stem Cells Overexpressed by Transcription Factor Combinations

[0046] The monoclonal induced pluripotent stem cells identified by pluripotency were expanded and cultured, and the induced pluripotent stem cells in the logarithmic growth phase were taken, digested and counted, seeded in a well plate at 5x104 power / ml, and mixed with MOI=100 Induced pluripotent stem cells were infected with transcription factor lentivirus, and the overexpression of exogenous TF gene was verified by q-PCR. The sequences of primers used to detect transcription factors are shown in Table 1.

[0047] The inventor selected six transcription factors (transcription factor Runx1, transcription factor Tcf1, transcription factor Id2, transcription factor Rorγt, transcription factor Batf3 and transcription factor Nfil3), and found that these six transcription factors were overexpressed in the induced pluripotent stem cells. After expression, the induce...

Embodiment 3

[0055] Example 3. Differentiation of induced pluripotent stem cells overexpressed in combination of transcription factors into lymphoid tissue induced cells

[0056] (1) Digest and resuspend the cells in the first differentiation medium (15% fetal bovine serum + IMEM + 1% glutamine + 50ug / ml ascorbic acid + 5ng / ml bone morphogenetic protein 4) to a cell density of 1x10 5 / ml, 20ul / drop was cultured for 2.5 days by hanging drop culture method to prepare embryoid bodies (embryonic body, EB).

[0057] (2) EBs of D2.5 were collected and transferred to well plates pre-coated with 0.1% gelatin. In the second differentiation medium (15% FBS + IMEM + 1% glutamine + 50 ug / ml ascorbic acid + 5 ng / ml bone morphogenetic protein 4 + 5 ng / ml vascular endothelial growth factor) continue to culture until the sixth day.

[0058] (3) The cells differentiated to D6 were digested with 0.25% trypsin-EDTA, collected and centrifuged, added to OP9-DL1 feeder cells for co-cultivation, and cultured in...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a method for directional differentiation of induced pluripotent stem cells into lymphoid tissue induced cells. At least one of a transcription factor Runx1, a transcription factor Tcf1, a transcription factor Id2, a transcription factor Rorgamma t, a transcription factor Batf3 and a transcription factor Nfil3 is overexpressed in the induced pluripotent stem cells. The induced pluripotent stem cell line is constructed by utilizing a stem cell technology, so that the construction requirement of transcription factor gene reporter mice of lymphatic progenitor cells in different stages is met, the repeatability of differentiation is improved by stably overexpressing a specific transcription factor combination, and the functional integrity of the lymphatic tissue induced cells obtained by induction is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for inducing directional differentiation of pluripotent stem cells into lymphoid tissue inducing cells. Background technique [0002] Induced Pluripotent Stem Cells (iPSCs) is a milestone breakthrough in the field of stem cell research in recent years. It is a pluripotent cell similar to ESCs obtained by ectopically expressing several key nuclear transcription factors related to the maintenance of pluripotency of embryonic stem cells (EmbryonicStem Cells, ESCs), which can be reprogrammed in vivo. All tissue cells differentiated into three germ layers inside and outside. iPSCs reprogrammed from somatic cells adds a new way to obtain pluripotent stem cells consistent with the patient's own genetic background, and no longer uses human early embryos and oocytes, so the ethical debate will subside. The dilemma of lack of oocytes and complex techniques in transpl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N15/867C12N5/10
CPCC12N5/0634C12N15/86C12N5/0696C07K14/47C12N2506/45C12N2501/998C12N2500/32C12N2500/38C12N2501/155C12N2501/165C12N2501/2303C12N2501/2306C12N2501/125C12N2740/15043C12N2800/107C12N2510/00
Inventor 宋尔卫苏士成陈惠萍
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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