Pluripotent stem cells directional differentiation method

Abstract

The invention discloses a pluripotent stem cells directional differentiation method. The method is characterized in that pluripotent stem cells are cultured under cell culture environment of non exogenous hematopoietic cytokines, non serum, non matrix cells and clear component, after embryoid is formed, a vascular endothelial growth factor and bone morphogenetic protein 4 are added, the embryoid is attached on the surface spread with collagen IV, mesoderm differentiation is induced, and hemopoietic progenitor cells and blood corpuscles are generated. The cell culture system with determined component enables directional differentiation on the pluripotent stem cells, has the advantages of low cost, simple operation, high repeatability and ordered differentiation process, can massively generate hemopoietic progenitor cells and blood corpuscles, and has important meaning for researching a blood growth process mechanism.

Description

technical field

[0001] The invention belongs to the technical field of cell culture and differentiation methods, more specifically, the invention relates to a method for directional differentiation of human pluripotent stem cells under the culture conditions of no exogenous hematopoietic cytokines, no serum, no stromal cells, and clear components. method. Background technique

[0002] Directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides an opportunity to obtain clinical-grade therapeutic hematopoietic progenitors and mature blood cells on a large scale, and provides conditions for functional studies in early hematopoietic development in humans.

[0003] Usually, most culture systems using hPSCs for directed hematopoietic development use mouse cell lines OP9, mAGM-S3, S17, and MS-5 trophoblast cells for co-culture, and these co-culture systems require the use of fetal bovine serum to maintain mouse stromal cells Grow and achieve differentiation ...

Examples

Embodiment 1

[0045] (1) Human pluripotent stem cells (hPSCs) were routinely cultured in serum-free mTeSR1 medium lined with Matrigel. After hPSCs were passaged at short intervals for 3–5 times, cells in the exponential growth phase were trypsinized into single-cell suspensions. After centrifugation, the cells were resuspended with mTeSR1 medium containing 1 μM Thiazovivin, and the single cell suspension was added to the AggreWell culture plate to form EBs. Each AgreeWell400 culture well had one million cells. Each EB contains 200-500 cells.

[0046] (2) EBs were cultured for 24 hours under standard hPSCs culture conditions (5% carbon dioxide, 21% oxygen, 37°C, humidified environment), and then the EBs were pipetted out carefully, and hVEGF containing 50ng / mL 165 , 2ng / mL hBMP4 and 10μM Thiazovivin mTeSR1 culture medium suspended, added to the mouse or human collagen IV (0.5μg / cm 2 ) on plastic culture plates. The culture density of EBs is 15-20 per square centimeter. Addition of Activi...

Embodiment 2

[0051] This example provides a method for expanding the scale of cell culture. The steps are the same as in Example 1, except that the cells are treated with trypsin during the second to fourth days of differentiation, and then re-plated on the culture plate coated with collagen IV or On the petri dish, incubate for 1 h under the standard culture environment. Then gently pipet and aspirate the cells that are still in suspension or in a semi-adsorbed state to remove residual undifferentiated hPSCs. The adsorbed cell part can continue to be cultured in the original culture dish according to the above conditions, or be seeded in a new collagen IV-coated culture dish at a density of 10,000 cells per square centimeter. The method of this embodiment can expand the scale of the whole cell culture about ten times.

experiment example 1

[0053] To analyze blood progenitor cells generated during differentiation, whole cells or sorted cells at different stages of differentiation were added to a culture medium containing the human cytokines SCF, G-CSF, GM-CSF, IL-3, IL-6, and EPO. Colony analysis was performed in serum-free methylcellulose medium. The earliest blood progenitors appear on the fourth day of differentiation. Most blood cells with blood colony forming ability are present in the suspension cell fraction. Essentially all blood progenitors are present on CD43 + cell population (see Figure 6 ). CD43 + cells and CD43 - Compared with cells, many blood cell-related genes are highly expressed (see Figure 7 ). In the early stages of differentiation, erythroid progenitors and multipotent progenitors (CFU-E, BFU-E and CFU-Mix) are the main hematopoietic progenitors generated. At the later stage of differentiation (differentiation day 14 to 16), the multipotent progenitor cells were well maintained due...

technical field

[0001] The invention belongs to the technical field of cell culture and differentiation methods, more specifically, the invention relates to a method for directional differentiation of human pluripotent stem cells under the culture conditions of no exogenous hematopoietic cytokines, no serum, no stromal cells, and clear components. method. Background technique

[0002] Directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides an opportunity to obtain clinical-grade therapeutic hematopoietic progenitors and mature blood cells on a large scale, and provides conditions for functional studies in early hematopoietic development in humans.

[0003] Usually, most culture systems using hPSCs for directed hematopoietic development use mouse cell lines OP9, mAGM-S3, S17, and MS-5 trophoblast cells for co-culture, and these co-culture systems require the use of fetal bovine serum to maintain mouse stromal cells Grow and achieve differentiation ...

Examples

Embodiment 1

[0045] (1) Human pluripotent stem cells (hPSCs) were routinely cultured in serum-free mTeSR1 medium lined with Matrigel. After hPSCs were passaged at short intervals for 3–5 times, cells in the exponential growth phase were trypsinized into single-cell suspensions. After centrifugation, the cells were resuspended with mTeSR1 medium containing 1 μM Thiazovivin, and the single cell suspension was added to the AggreWell culture plate to form EBs. Each AgreeWell400 culture well had one million cells. Each EB contains 200-500 cells.

[0046] (2) EBs were cultured for 24 hours under standard hPSCs culture conditions (5% carbon dioxide, 21% oxygen, 37°C, humidified environment), and then the EBs were pipetted out carefully, and hVEGF containing 50ng / mL 165 , 2ng / mL hBMP4 and 10μM Thiazovivin mTeSR1 culture medium suspended, added to the mouse or human collagen IV (0.5μg / cm 2 ) on plastic culture plates. The culture density of EBs is 15-20 per square centimeter. Addition of Activi...

Embodiment 2

[0051] This example provides a method for expanding the scale of cell culture. The steps are the same as in Example 1, except that the cells are treated with trypsin during the second to fourth days of differentiation, and then re-plated on the culture plate coated with collagen IV or On the petri dish, incubate for 1 h under the standard culture environment. Then gently pipet and aspirate the cells that are still in suspension or in a semi-adsorbed state to remove residual undifferentiated hPSCs. The adsorbed cell part can continue to be cultured in the original culture dish according to the above conditions, or be seeded in a new collagen IV-coated culture dish at a density of 10,000 cells per square centimeter. The method of this embodiment can expand the scale of the whole cell culture about ten times.

experiment example 1

[0053] To analyze blood progenitor cells generated during differentiation, whole cells or sorted cells at different stages of differentiation were added to a culture medium containing the human cytokines SCF, G-CSF, GM-CSF, IL-3, IL-6, and EPO. Colony analysis was performed in serum-free methylcellulose medium. The earliest blood progenitors appear on the fourth day of differentiation. Most blood cells with blood colony forming ability are present in the suspension cell fraction. Essentially all blood progenitors are present on CD43 + cell population (see Figure 6 ). CD43 + cells and CD43 - Compared with cells, many blood cell-related genes are highly expressed (see Figure 7 ). In the early stages of differentiation, erythroid progenitors and multipotent progenitors (CFU-E, BFU-E and CFU-Mix) are the main hematopoietic progenitors generated. At the later stage of differentiation (differentiation day 14 to 16), the multipotent progenitor cells were well maintained due...

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