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Phagocytosis model of calcium oxalate monohydrate crystals generated using human induced pluripotent stem cell-derived macrophages

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Abstract

Macrophages play a role in nephrolithiasis, offering the possibility of developing macrophage-mediated preventive therapies. To establish a system for screening drugs that could prevent the formation of kidney stones, we aimed to develop a model using human induced pluripotent stem cell (iPSC)-derived macrophages to study phagocytosis of calcium oxalate monohydrate (COM) crystals. Human iPSCs (201B7) were cultured. CD14+ monocytes were recovered using a stepwise process that involved the use of growth factors and cytokines. These cells were then allowed to differentiate into M1 and M2 macrophages. The macrophages were co-cultured with COM crystals and used in the phagocytosis experiments. Live cell imaging and polarized light observation via super-resolution microscopy were used to visualize phagocytosis. Localization of phagocytosed COM crystals was observed using transmission electron microscopy. Intracellular fluorescence intensity was measured using imaging cytometry to quantify phagocytosis. Human iPSCs successfully differentiated into M1 and M2 macrophages. M1 macrophages adhered to the culture plate and moved COM crystals from the periphery to cell center over time, whereas M2 macrophages did not adhere to the culture plate and actively phagocytosed the surrounding COM crystals. Fluorescence assessment over a 24-h period showed that M2 macrophages exhibited higher intracellular fluorescence intensity (5.65-times higher than that of M1 macrophages at 4.5 h) and maintained this advantage for 18 h. This study revealed that human iPSC-derived macrophages have the ability to phagocytose COM crystals, presenting a new approach for studying urinary stone formation and highlighting the potential of iPSC-derived macrophages as a tool to screen nephrolithiasis-related drugs.

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Data Availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

Not applicable.

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Acknowledgments

We thank Ms. Naomi Kasuga and Ms. Yoko Ito for their assistance with the experimental studies. This study was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (20K09562) and a JUA Research Grant from the Japanese Urological Association (27th, 2018).

Funding

This study was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (16K15692, 19H03791, 20K09562, 21K09405, 21K07803, 22H00486, 23H03039), Research Grand from SUZUKI MEMORIAL FOUNDATION (2018), TOYOAKI SCHOLARSHIP FOUNDATION (2023) and a JUA Research Grant from the Japanese Urological Association (27th, 2018).

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Author notes

  1. Tomoki Okada, Atsushi Okada have equally contribution.

Authors and Affiliations

  1. Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan

    Tomoki Okada, Atsushi Okada, Rei Unno, Kazumi Taguchi, Shuzo Hamamoto, Ryosuke Ando & Takahiro Yasui

  2. Department of Pathobiology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan

    Hiromasa Aoki

  3. Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan

    Daichi Onozato, Tadahiro Hashita, Takahiro Iwao & Tamihide Matsunaga

  4. Department of Urology, Nagoya City East Medical Center, Nagoya, Aichi, Japan

    Taiki Kato & Teruaki Sugino

  5. Core Laboratory, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan

    Hiroshi Takase

  6. Department of Medical Technology, Yokkaichi Nursing and Medical Technology school of Nursing and Medical Care, Yokkaichi, Mie, Japan

    Shigeru Ohshima

  7. Department of Cell Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan

    Issei S. Shimada

Authors
  1. Tomoki Okada
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  2. Atsushi Okada
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Contributions

Tomoki Okada and Atsushi Okada performed the experiments and wrote the manuscript as equal contributors. Hiromasa Aoki, Daichi Onozato, Taiki Kato, Hiroshi Takase, Shigeru Ohshima, Teruaki Sugino, Rei Unno, Kazumi Taguchi, Shuzo Hamamoto, and Ryosuke Ando assisted with the experiments and confirmed the results. Issei S Shimada, Tadahiro Hashita, Takahiro Iwao, Tamihide Matsunaga, and Takahiro Yasui revised the manuscript. All authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to Atsushi Okada.

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Okada, T., Okada, A., Aoki, H. et al. Phagocytosis model of calcium oxalate monohydrate crystals generated using human induced pluripotent stem cell-derived macrophages. Urolithiasis 52, 51 (2024). https://doi.org/10.1007/s00240-024-01553-8

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