A kind of preparation method and application of human hematopoietic progenitor cells with B lineage differentiation potential

A technology of hematopoietic progenitor cells and differentiation potential, which is applied in the field of preparation of human hematopoietic progenitor cells, can solve the problems of undetectable antibodies, lack of B cells, and short time of B cells, and achieves the effect of improving induction efficiency, high application value and large quantity.

Active Publication Date: 2022-07-19
BEIJING INST FOR STEM CELL & REGENERATIVE MEDICINE
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AI Technical Summary

Problems solved by technology

But there are the following problems: first, B cells exist in the body for a short time, and at the same time, the secreted antibodies cannot be detected after 6 to 8 weeks after transplantation (Potocnik, A J et al. Reconstitution of B cell subsets in Rag deficient mice by transplantation of in vitro differentiated embryonic stem cells. Immunology letters vol. 57,1-3 (1997): 131-7); second, B2 cells that are more important for adaptive humoral immune responses cannot be obtained (Lin, Yang et al. Long -Term Engraftment of ESC-Derived B-1 Progenitor Cells SupportsHSC-Independent Lymphopoiesis. Stem cell reports vol. 12,3 (2019):572-583)
The above studies have shown that there are certain defects in the system of in vitro induction of B progenitor cells by pluripotent stem cells and in vivo B cell regeneration after B progenitor cell transplantation. It is speculated that the lack of specific transcription factors may lead to certain defects in the regenerated B lymphocyte lineage
However, there is no report about inducing the differentiation of human pluripotent stem cells in vitro to produce hematopoietic progenitor cells with B-lineage differentiation potential (that is, B-lineage seed cells), and the regeneration of human B cells after transplantation

Method used

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  • A kind of preparation method and application of human hematopoietic progenitor cells with B lineage differentiation potential
  • A kind of preparation method and application of human hematopoietic progenitor cells with B lineage differentiation potential
  • A kind of preparation method and application of human hematopoietic progenitor cells with B lineage differentiation potential

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Embodiment 1

[0122] Example 1 Preparation of human pluripotent stem cells inducibly expressing exogenous RUNX1, HOXA9 and LHX2 genes

[0123] In this example, based on the CRISPR / Cas9 system, an inducible expression sequence was knocked into the AAVS1 site of human pluripotent stem cells (hPSC) by electroporation, and the knock-in sequence included RUNX1-p2a-HOXA9-t2a-LHX2 The tandem sequence, the fluorescent protein gene GFP and the puromycin resistance gene sequence for resistance screening, the schematic diagram is as follows Figure 1A shown.

[0124] The sgRNA and targeting vector were electroporated into hPSCs. After 20 h, hPSC medium containing puromycin (1 μM) was added, and the medium was changed every day. Wherein, the target sequence corresponding to the used sgRNA is shown in SEQ ID No.1.

[0125] SEQ ID No. 1: 5'-gtcaccaatcctgtccctagtgg-3' (hAAVS1-sgRNA).

[0126] After 10 days of selection with puromycin, culture, expansion and passage were carried out in puromycin-free med...

Embodiment 2

[0134] Example 2 RUNX1, HOXA9 and LHX2 triple gene-modified human pluripotent stem cells induce differentiation to produce hematopoietic progenitor cells

[0135] The flow chart of the induction and differentiation of human pluripotent stem cells to generate hematopoietic progenitor cells (HPC) is as follows Figure 2A shown.

[0136] Using a monolayer culture-induced differentiation system based on stromal cell co-culture, the induced differentiation process is divided into three stages:

[0137] (1) The first stage is to induce human pluripotent stem cells to generate mesoderm cells (D0 to D2), Figure 2B Brightfield images of cells during induction of mesoderm cells are shown:

[0138] Culture in D0 medium and D1 medium respectively;

[0139] D0 medium was formulated as: 15 ng / mL hbFGF (Peprotech), 20 ng / mL rhActivin A (Peprotech), 30 ng / mL rhBMP4 (R&D), 4 μM CHIR99021 (Selleck) and 6 μM LY294002 (Selleck), balance It is TeSR-E6 basal medium (STEMCELL Technologies);

...

Embodiment 3

[0150] Example 3 Transplantation of iRUNX1-p2a-HOXA9-t2a-LHX2-hPSC-derived hematopoietic progenitor cells (B lineage seed cells) into immunodeficient mice and detection of B cell regeneration

[0151] Sort D16 to get CD34 + Cells were transplanted into immunodeficient NCG mice (Jicui Yaokang) with sublethal irradiation (2-3 Gy) by intravenous injection into the inner canthus at a dose of 1 million per mouse.

[0152] At 6, 8 and 10 weeks after transplantation, the peripheral blood of the recipient mice was collected by tail vein blood for flow analysis. The staining scheme was as follows: GFP, mTER119-PerCP / Cy5.5, hCD45-PE / Cy7, hCD19 - BV785, hCD33-PE, hCD3 / 4 / 8-APC and DAPI (all of the above antibodies were purchased from Biolegend). Data were analyzed using Flowjo software.

[0153] The result is as image 3 As shown in the figure, iRUNX1-p2a-HOXA9-t2a-LHX2-HPC can be used as B lineage seed cells to regenerate CD19 in recipient mice + B cells.

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Abstract

The invention provides a preparation method and application of human hematopoietic progenitor cells with B lineage differentiation potential. The preparation method comprises: introducing three transcription factors RUNX1, HOXA9 and LHX2 into a human pluripotent stem cell induction differentiation system simultaneously or separately The cells of the human pluripotent stem cell induced differentiation system include human pluripotent stem cells, mesoderm cells, hematopoietic endothelial cells or Any one or a combination of at least two of the hematopoietic progenitor cells; the method for inducing differentiation includes a method for inducing differentiation by monolayer culture based on co-culture of stromal cells or a method for inducing differentiation by three-dimensional culture of organoids. The preparation method has good induction and differentiation efficiency, and the prepared cells have good activity and high differentiation ability, and have important application value.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to a preparation method and application of a human hematopoietic progenitor cell with B lineage differentiation potential. Background technique [0002] B cells are the key cellular components of the body's humoral immune system. Patients with low or defective B cell function, such as the elderly or patients with underlying diseases, will lead to impaired humoral immunity and even severe pathogen infection. In addition, genetically engineered B cells (engineering B cells) have considerable application prospects in the field of clinical anti-infection, anti-tumor and disease treatment. [0003] However, clinically, drugs such as B cells and B cell-derived gamma globulin preparations are derived from human peripheral blood or bone marrow banks, with limited resources, long preparation time and high cost. Pluripotent stem cells (PSCs) are a class of cells with unlimited...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12C12N5/10C12N5/0789A61K35/28A61P35/00A61P37/02A61P31/00A61P7/04A61P43/00
CPCC12N15/85C12N15/907C07K14/4702C12N5/0696C12N5/0647A61K35/28A61P35/00A61P37/02A61P31/00A61P7/04A61P43/00C12N2510/00C12N2506/45C12N2501/60C12N2501/115C12N2501/155C12N2501/727C12N2501/415C12N2500/38C12N2501/165C12N2501/125C12N2501/2303C12N2501/22C12N2500/30C12N2501/145C12N2501/2311C12N2501/105C12N2501/15
Inventor 王金勇张琪夏成祥王瑶王童洁翁启童林云轻朱艳平
Owner BEIJING INST FOR STEM CELL & REGENERATIVE MEDICINE
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