Method for differentiating human multipotential stem cells into hematopoietic stem cells and culture additive
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A technology for hematopoietic stem cells and additives, which is applied in cell culture active agents, artificially induced pluripotent cells, biochemical equipment and methods, etc. stability issues
Active Publication Date: 2018-07-20
TSINGHUA UNIV
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These methods also have some defects: the embryoid body method generally needs to consume a large number of pluripotent stem cells, and the inconsistency of their differentiation stages leads to their generally low differentiation efficiency and time-consuming; the efficiency of the stromal cell co-culture method is unstable, and animal sources Element
For example, co-cultivation with mouse bone marrow stromal cells OP9 cells will introduce mouse-derived components, which will bring safety hazards to the clinical application of induced differentiation of hematopoietic stem cells
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Embodiment 1
[0129] Example 1. Differentiation of human pluripotent stem cells to hematopoietic stem cells
[0130] Two types of human pluripotent stem cells were used in this example, namely H1 cells and CD34-iPSC cells.
[0131] 1. Inoculate human pluripotent stem cells in a 6-well plate (2.5×10 per well 5 cells) were cultured in TeSR-E8 medium at 37°C until the cell confluency was 70%-80%.
[0132] 2. After completing step 1, take the six-well plate, suck off the culture supernatant, and add PBS buffer preheated to 37°C to wash twice. At this point, the morphology of H1 cells and CD34-iPSC cells is shown in figure 1 (Scale bar 100 μm).
[0133] 3. After completing step 2, take the six-well plate, add 1ml of cell digestion solution Accutase to each well, let it stand at 37°C for 3-5min, then add an appropriate amount of RPMI 1640 basal culture medium to stop the digestion, and collect the cells by centrifugation.
[0134] 4. Inoculate the cells collected in step 3 on a petri dish (th...
Embodiment 2
[0143] Example 2, Detection of cells in the process of differentiation of pluripotent stem cells to hematopoietic stem cells
[0144] 1. Detection of cell B
[0145] 1, get the cell B that step 8 obtains in embodiment 1, adopt the PBS damping fluid resuspended cell that contains 5% (v / v) fetal bovine serum to obtain cell suspension (containing 1 * 10 5 cells).
[0146] 2. Add PE-labeled FLK1 antibody to the cell suspension in step 1 (set PE-labeled IgG antibody instead of PE-labeled FLK1 antibody as a negative control), incubate at room temperature in the dark for 20 minutes (during this period, mix well every 5 minutes) once); then washed twice with PBS buffer containing 5% (v / v) fetal bovine serum, and centrifuged to collect the cells.
[0147] 3. After completing step 2, use 500 μL of PBS buffer containing 5% (v / v) fetal bovine serum to resuspend the cells, and use flow cytometry to detect.
[0148] For the results of the detection of mesoderm precursor cells obtained fr...
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Abstract
The invention discloses a method for differentiating human multipotential stem cells into hematopoietic stem cells and a culture additive. The invention develops a culture solution capable of greatlyimproving the efficiency of differentiating human multipotential stem cells into hematopoietic stem cells. The invention also provides a novel method for differentiating human multipotential stem cells into hematopoietic stem cells. The hematopoietic stem cells can be obtained by adopting the preparation method provided by the invention, and compared with traditional matrix cell co-culture methodand embryoid body culture method, the doubly positive hematopoietic stem cells capable of expressing CD34 and CD343 can be efficiently obtained through the method, and the chemical components are definite and free of animal-derived components, so that the safety of cell preparation is greatly improved, and the method has the characteristics of low time consumption, high differentiation efficiency,relatively low cost and the like. The artificial hematopoietic stem cells can be produced in large scale by adopting the preparation method provided by the invention, the method is stable in qualityand high in safety, and a large number of cell sources are provided for tissue engineering, research and development of drugs and cell therapy.
Description
technical field [0001] The invention relates to a method for differentiating human pluripotent stem cells into hematopoietic stem cells and a culture additive. Background technique [0002] Human pluripotent stem cells include human embryonic stem cells and human induced pluripotent stem cells, which can differentiate into various tissues in the human body. They can be used to make disease models, conduct drug toxicity tests, and replace damaged and diseased cells through cell transplantation to promote body trauma. Repair and cure diseases. Hematopoietic stem cells exist in the human body for life and can differentiate into various cells of the blood system, including red blood cells, granulocytes, macrophages, monocytes, microglia, dendritic cells, B-lymphocytes, T-lymphocytes, NK-lymphocytes are of great value in the clinical treatment of blood diseases and cancer. [0003] At present, the main ways to induce the differentiation of human pluripotent stem cells into hema...
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