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In-vitro preparation of eosinophilic granulocytes from human pluripotent stem cells

A technology of eosinophils and pluripotent stem cells, applied in the field of stem cell differentiation, can solve the problem of limiting the anti-tumor effect of eosinophils

Pending Publication Date: 2021-10-12
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These features limit the antitumor role of eosinophils in cancer therapy

Method used

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  • In-vitro preparation of eosinophilic granulocytes from human pluripotent stem cells
  • In-vitro preparation of eosinophilic granulocytes from human pluripotent stem cells
  • In-vitro preparation of eosinophilic granulocytes from human pluripotent stem cells

Examples

Experimental program
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Embodiment 1

[0166] Example 1 Obtaining Eosinophils Efficiently Differentiated from Human Embryonic Stem Cells (hESCs)

[0167] Previously, we studied the generation of hematopoietic cells from hPSCs (Wang et al., 2012). In the present invention, to generate eosinophils from hESCs, we developed a novel differentiation method by improving a previously established stepwise differentiation strategy by exposure to specific differentiation media to stepwise generate mesodermal, hematopoietic cells. Endothelial cells and hematopoietic progenitor cells (see figure 1 A and "Experimental Materials and Methods" section). After day 12, interleukin 3 (IL-3) and interleukin 5 (IL-5), which support eosinophil development and survival (Reichman et al., 2016), were introduced to induce eosinophil differentiation and maturation (Method and parameters refer to the "In vitro directed differentiation of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) into eosinophils" sec...

Embodiment 2

[0170] Example 2 Transcription analysis of hESC-derived eosinophils

[0171] We performed RNA-sequencing on parental H1 cells as well as differentiated cells harvested at D12 (E0), D16 (E4), D20 (E8), D24 (E12), D28 (E16), and D32 (E20). The data revealed that the differentiation process undergoes a transition from PSCs to eosinophils with a stage-specific clustering pattern ( figure 2 A and B). During hematopoietic induction, pluripotent genes were downregulated, such as NANOG and POU5F1; while HE-associated genes CD34 and CD45 were upregulated during hematopoietic induction and downregulated during eosinophil induction ( figure 2 B). Key developmental regulators of eosinophils, such as transcription factors CEBPA, CEBPE, SPI1, and GATA1 (Reichman et al., 2016) and receptors IL5RA, CSF2RB, ADGRE1, and CD52 (Davis and Rothenberg, 2014) ) expression gradually increases during eosinophilia; notably, expression of the maturation marker SIGLEC8 (Rosenberg et al., 2013) is ind...

Embodiment 3

[0174] Example 3 Generation of Eosinophils by Efficient Differentiation of Human iPSCs

[0175] To investigate whether our protocol could be applied to other hPSC lines, we used human induced pluripotent stem cells (iPSCs) for eosinophil differentiation ( Image 6 A). iPSCs show faster differentiation of hematopoietic progenitors than H1 cells, so we replaced the medium with eosinophil induction medium on day 8 (E0) to induce eosinophils (see "Experimental Materials and Methods "part)( Image 6 A). to EPX + Cell tracking revealed that eosinophils begin to produce EPX 4 days after induction + cells, whose number gradually increased and reached 100% at E20, yielding nearly 6.00 × 10 per culture plate 7 cells expressing Siglec-8, CD69 and CD11b ( Image 6 B-E). iPSC-derived EPX + The percentage and number of cells plateaued at day 28 (E20).

[0176] Next, we investigated the gene expression profile of human iPSC-derived eosinophils using RNA-seq and real-time qPCR. Cell...

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Abstract

The invention discloses eosinophilic granulocytes and a preparation method and application thereof. The invention provides a method for preparing eosinophilic granulocytes, which comprises the following steps: (1) culturing pluripotent stem cells in a mesoderm induction culture medium to obtain mesoderm cells; (2) culturing the mesoderm cells obtained in the step (1) in a hematopoietic endothelial induction culture medium to obtain hematopoietic endothelial cells which are CD34+CDH5+cells; (3) culturing the hematopoietic endothelial cells obtained in the step (2) in a hematopoietic progenitor cell induction culture medium to obtain hematopoietic progenitor cells which are CD34+CD45+cells; and (4) culturing the hematopoietic progenitor cells obtained in the step (3) in an eosinophilic granulocyte induction culture medium to obtain the eosinophilic granulocytes. In the preparation method disclosed by the invention, the components of the culture mediums are clear, the induction process is simple, and a large number of functional eosinophilic granulocytes can be efficiently prepared.

Description

technical field [0001] The invention belongs to the technical field of stem cell differentiation, and more specifically, the invention relates to a method for inducing pluripotent stem cells to prepare eosinophils. Background technique [0002] The successful migration and efficient infiltration of immune effector cells into tumors is a prerequisite for tumor immunotherapy to produce anti-tumor efficacy ( Guedan et al., 2019; Miller and Lanier, 2019 ; Newick et al.,2017 ). At present, the efficacy of various immunotherapies for treating solid tumors is severely limited by the difficulty of immune cells infiltrating into solid tumors ( Grosser et al., 2019; Habif et al., 2019 ; Rafiq et al.,2019 ). Although there have been a lot of attempts, it is still difficult to promote the efficient infiltration of immune effector cells into the tumor ( Harrington et al.,2019 ; Li et al.,2019 ; Morel et al.,2020 ; Nayyar et al.,2019 ; Park et al.,2012 ). Therefore, the...

Claims

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Application Information

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IPC IPC(8): C12N5/0787C12Q1/6888C12Q1/04A61K35/15A61P35/00
CPCC12N5/0642C12Q1/6888A61K35/15A61P35/00C12N2501/2303C12N2501/2305C12N2501/155C12N2501/165C12N2501/115C12N2501/15C12N2501/125C12N2501/22C12N2506/02C12N2506/45C12Q2600/158
Inventor 邓宏魁王承艳来威锋谢皇帆
Owner PEKING UNIV