Int J Stem Cells.  2023 Feb;16(1):108-116. 10.15283/ijsc22150.

Lymphoid Lineage γδ T Cells Were Successfully Generated from Human Pluripotent Stem Cells via Hemogenic Endothelium

• Jeon SB
• Han AR
• Choi YB
• Lee AR
• Lee JY

• KMID: 2539625
• DOI: http://doi.org/10.15283/ijsc22150

Abstract

γδ T cells are a rare and unique prototype of T cells that share properties with natural killer cells in secondary lymphoid organs. Although many studies have revealed the function and importance of adult-derived γδ T cells in cancer biology and regenerative medicine, the low numbers of these cells hamper their application as therapeutic cell sources in the clinic. To solve this problem, pluripotent stem cell-derived γδ T cells are considered alternative cell sources; however, few studies have reported the generation of human pluripotent stem cell-derived γδ T cells. In the present study, we investigated whether lymphoid lineage γδ T cells were successfully generated from human pluripotent stem cells via hemogenic endothelium under defined culture conditions. Our results revealed that pluripotent stem cells successfully generated γδ T cells with an overall increase in transcriptional activity of lymphoid lineage genes and cytolytic factors, indicating the importance of the optimization of culture conditions in generating lymphoid lineage γδ T cells. We uncovered an initial step in differentiating γδ T cells that could be applied to basic and translational investigations in the field of cancer biology. Based on our result, we will develop an appropriate method to purify γδ T cells with functionality and it helpful for the study of basic mechanism of γδ T cells in pathophysiologic condition as well as clinic application.

Keyword

γδ T cells; Hemogenic endothelium; Embryonic stem cells; Lymphoid lineage transcription factors

Figure

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  Fig. 1 Schematic for generating γδ T cells from PSCs via HE according to developmental biology. There are four steps to differentiate into γδ T cells: stage I, yolk sac blood island to mesodermal lineage; stage II, HE in aorta-gonad mesonephros; stage III, pre γδ T cells development in fetal liver; stage IV, generation of γδ T cells and growth in fetal liver. Diverse cytokines were added in each step to optimize the generation of γδ T cells.

• 

  Fig. 2 HE from PSCs was stably generated at days 5 and 6. (A) Representative morphology of PSC-derived HE according to BMP4. At days 5 and 6, yellow lines depict HE territory. Magnification 20×. (B) At day 6, the TIE-2 and CD34 marker for HE was expressed in adherent cells. TIE-2 (Green), CD34 (red), and DAPI (blue) for nuclear staining are demarcated in HE. Magnification 20× and 40×. (C) Percentage of TIE-2 and CD34 cells in differentiated cells at days 5 and 6. Images are representative of at least two independent experiments. (D) The percentage of CXCR4-CD73-cells in TIE-2＋ cells and CD34＋ cells in differentiated cells at days 5 and 6. Images from three independent experiments are shown.

• 

  Fig. 3 γδ T cells from HE were successfully generated under defined culture conditions. (A) Representative morphology of γδ T cells from HE until day 39. Wright and Giemsa staining showed lymphoid lineage cells. Magnification 20×. (B) Flow cytometry results revealed that markers for γδ T cells were expressed in differentiated cells at days 26 and 30. The numbers of γδ T cells were significantly increased. Results are shown as the means±SEM from at least two independent experiments *p<0.05, **p<0.01. (C) Immunochemistry findings for CD56 and NKG2D expression in differentiated cells, including HE and floating cells, on day 26. Magnification 20×. (D) FACS results showed no difference in the percentages of Vδ2＋CD3＋ and Vγ9＋CD3＋ cells in HE and floating cells, respectively. Results are from at least three independent experiments. (E) FACS results revealed that the numbers of CD3, TCR Vδ2＋, TCR Vγ9＋, and Vδ2＋Vγ9＋ in CD3 cells gradually increased over time in the floating cells than those in HE. Results are shown as the means±SEM from at least two independent experiments. *p<0.05. (F) Genes according to each developing steps were determined in differentiated cells. mRNA expression of representative marker genes was determined through qRT-PCR. Results are shown as the means±SEM.

Reference

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