•  
  •  
 

ORCID ID

Samuel Febrian Wijaya : 0000-0002-6266-7236

Retno Lestari : 0000-0003-2006-0214

Inna Rahmawati : 0000-0001-6216-7496

Imelda Rosalyn Sianipar : 0000-0002-1438-096X

Robby Nuraditya : -

Iqbal Fasha : -

Gita Pratama : 0000-0001-5626-1283

Radiana Dhewayani Antarianto : 0000-0002-8578-7505

Abstract

Background: Natural killer (NK) cells originate from the differentiation of hematopoietic stem cells (HSCs) in the common lymphoid progenitor pathway, and HSCs can be obtained from umbilical cord blood (UCB). Comparative studies of NK cell differentiation between cultured and freshly isolated HSCs are important in the development of NK cell therapy for cancer. This study aimed to compare the maturation stages of NK cell differentiation between cultured and newly isolated HSC samples using interleukin-2 in the absence of feeder cells.

Methods: Differentiation cultures were divided into two groups according to HSC source. Giemsa staining and flow cytometry were performed to determine the maturation stages and the presence of NKp46 receptors, respectively.

Results: Giemsa staining revealed that the cultured HSC samples produce a higher number and more mature (stage 5) NK cells than the freshly isolated HSC samples. Flow cytometry showed that the NKp46 mean fluorescence intensity significantly differed between the two samples, and a high level of NKp46 activation receptor was found in the isolated samples on day 35.

Conclusions: The cultured HSC samples could produce more mature NK cell populations than the freshly isolated HSCs, which will be beneficial for the therapy applications of NK cells derived from UCB HSCs.

References

  1. Yadav P, Vats R, Bano A, Bhardwaj R. Hematopoietic stem cells culture, expansion and differentiation: An insight into variable and available media. Int J Stem Cells. 2020;13:326–34.
  2. Khaddour K, Hana CK, Mewawalla P. Hematopoietic Stem Cell Transplantation. 2022 Jun 27. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan–.
  3. Freud AG, Yu J, Caligiuri MA. Human natural killer cell development in secondary lymphoid tissues. Semin Immunol. 2014;26:132–7.
  4. Björkström NK, Ljunggren HG, Michaëlsson J. Emerging insights into natural killer cells in human peripheral tissues. Nat Rev Immunol. 2016;16:310–20.
  5. Geiger TL, Sun JC. Development and maturation of natural killer cells. Curr Opin Immunol. 2016;39:82–9.
  6. Luevano M, Madrigal A, Saudemont A. Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy. Cell Mol Immunol. 2012;9:310–20.
  7. Wu Y, Tian Z, Wei H. Developmental and Functional control of natural killer cells by cytokines. Front Immunol. 2017;8:930.
  8. Dezell SA, Ahn YO, Spanholtz J, Wang H, Weeres M, Jackson S, et al. Natural killer cell differentiation from hematopoietic stem cells: A comparative analysis of heparin- and stromal cell-supported methods. Biol Blood Marrow Transplant. 2012;18:536–45.
  9. Liu S, Galat V, Galat Y, Lee YKA, Wainwright D, Wu J. NK cell-based cancer immunotherapy: From basic biology to clinical development. J Hematol Oncol. 2021;14:7.
  10. Abel AM, Yang C, Thakar MS, Malarkannan S. Natural killer cells: Development, maturation, and clinical utilization. Front Immunol. 2018;9:1869.
  11. Mizrahi O, Ish Shalom E, Baniyash M, Klieger Y. Quantitative flow cytometry: Concerns and recommendations in clinic and research. Cytometry B Clin Cytom. 2018;94:211–8.
  12. Eom S, Bise R, Kanade T. Detection of hematopoietic stem cells in microscopy images using a bank of ring filters. Proceedings of the 7th IEEE International Symposium on Biomedical Imaging: From Nano to Macro. 2010 Apr 14–17; Rotterdam, Netherlands.
  13. Shannon MJ, Mace EM. Natural killer cell integrins and their functions in tissue residency. Front Immunol. 2021;12:647358.
  14. Ullmo I, Koksal N, Ang HYK, Brady HJM. In vitro development of mouse and human nk cells from hematopoietic progenitor cells. Methods Mol Biol. 2022;2463:31–45.
  15. Koehl U, Brehm C, Huenecke S, Zimmermann SY, Kloess S, Bremm M, et al. Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol. Front Oncol. 2013;3:118.
  16. Luevano M, Domogala A, Blundell M, Jackson N, Pedroza-Pacheco I, Derniame S, et al. Frozen cord blood hematopoietic stem cells differentiate into higher numbers of functional natural killer cells in vitro than mobilized hematopoietic stem cells or freshly isolated cord blood hematopoietic stem cells. PLoS One. 2014;9:e87086.
  17. Yu H, Fehniger TA, Fuchshuber P, Thiel KS, Vivier E, Carson WE, et al. Flt3 ligand promotes the generation of a distinct CD34(+) human natural killer cell progenitor that responds to interleukin-15. Blood. 1998;92:3647–57.
  18. Domogala A, Blundell M, Thrasher A, Lowdell MW, Madrigal JA, Saudemont A. Natural killer cells differentiated in vitro from cord blood CD34+ cells are more advantageous for use as an immunotherapy than peripheral blood and cord blood natural killer cells. Cytotherapy. 2017;19:710–20.
  19. Del Zotto G, Antonini F, Pesce S, Moretta F, Moretta L, Marcenaro E. Comprehensive phenotyping of human PB NK cells by flow cytometry. Cytometry A. 2020;97:891–9.
  20. Mu YX, Zhao YX, Li BY, Bao HJ, Jiang H, Qi XL, et al. A simple method for in vitro preparation of natural killer cells from cord blood. BMC Biotechnol. 2019;19:80.
  21. Hussein BA, Hallner A, Wennström L, Brune M, Martner A, Hellstrand K, et al. Impact of NK cell activating receptor gene variants on receptor expression and outcome of immunotherapy in acute myeloid leukemia. Front Immunol. 2021;12:796072. Erratum in: Front Immunol. 2022;12:843461.
  22. Sinenko SA, Starkova TY, Kuzmin AA, Tomilin AN. Physiological Signaling functions of reactive oxygen species in stem cells: From flies to man. Front Cell Dev Biol. 2021;9:714370.
  23. Jabbar S, Mathews P, Kang Y. Emerging evidence of the significance of Thioredoxin-1 in hematopoietic stem cell aging. Antioxidants (Basel). 2022;11:1291.
  24. Degouve S, Tavares A, Viel S, Walzer T, Marçais A. NKp46-mediated Dicer1 inactivation results in defective NK-cell differentiation and effector functions in mice. Eur J Immunol. 2016;46:1902–11.
  25. Weidner CI, Walenda T, Lin Q, Wölfler MM, Denecke B, Costa IG, et al. Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture. Sci Rep. 2013;3:3372.
  26. Goni R, García P, Foissac S. The qPCR data statistical analysis. Integromics White Paper. 2009:1–9.
  27. Browne DJ, Brady JL, Waardenberg AJ, Loiseau C, Doolan DL. An analytically and diagnostically sensitive RNAextraction and RT-qPCR Protocol for peripheral blood mononuclear cells. Front Immunol. 2020;11:402.
  28. Granzin M, Wagner J, Köhl U, Cerwenka A, Huppert V, Ullrich E. Shaping of natural killer cell antitumor activity by ex vivo cultivation. Front Immunol. 2017;8:458.
  29. Sarvaria A, Jawdat D, Madrigal JA, Saudemont A. Umbilical cord blood natural killer cells, their characteristics, and potential clinical applications. Front Immunol. 2017;8:329.

Creative Commons License

Creative Commons Attribution-Share Alike 3.0 License
This work is licensed under a Creative Commons Attribution-Share Alike 3.0 License.

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.