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Establishment of an immortalized human erythroid cell line sustaining differentiation potential without inducible gene expression system

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Abstract

Ex vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential. Here, we report that erythroid cell lines established from human bone marrow using simple expression of HPV-E6/E7 are capable of normal erythroid differentiation, without turning gene expression off. Through simply changing cell culture conditions, a newly established cell line, Erythroid Line from Lund University (ELLU), is able to differentiate toward mature cells, including enucleated reticulocytes. ELLU is heterogeneous and, unexpectedly, clones expressing adult hemoglobin rapidly differentiate and produce fragile cells. Upon differentiation, other ELLU clones shift from fetal to adult hemoglobin expression, giving rise to more mature cells. Our findings propose that it is not necessary to employ gene induction systems to establish immortalized erythroid cell lines sustaining differentiation potential and describe novel cellular characteristics for desired functionally competent clones.

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References

  1. Zeuner A, Martelli F, Vaglio S, Federici G, Whitsett C, Migliaccio AR. Concise review: stem cell-derived erythrocytes as upcoming players in blood transfusion. Stem Cells. 2012;30:1587–96.

    Article  CAS  Google Scholar 

  2. Migliaccio AR, Masselli E, Varricchio L, Whitsett C. Ex-vivo expansion of red blood cells: how real for transfusion in humans? Blood Rev. 2012;26:81–95.

    Article  Google Scholar 

  3. Doorbar J, Egawa N, Griffin H, Kranjec C, Murakami I. Human papillomavirus molecular biology and disease association. Rev Med Virol. 2015;25(Suppl 1):2–23.

    Article  CAS  Google Scholar 

  4. Wong S, Keyvanfar K, Wan Z, Kajigaya S, Young NS, Zhi N. Establishment of an erythroid cell line from primary CD36+ erythroid progenitor cells. Exp Hematol. 2010;38:994–1005.

    Article  CAS  Google Scholar 

  5. Kurita R, Suda N, Sudo K, et al. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS ONE. 2013;8:e59890.

    Article  CAS  Google Scholar 

  6. Trakarnsanga K, Griffiths RE, Wilson MC, Blair A, Satchwell TJ, Meinders M, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat Commun. 2017;8:14750.

    Article  CAS  Google Scholar 

  7. Trakarnsanga K, Tipgomut C, Metheetrairut C, Wattanapanitch M, Khuhapinant A, Poldee S, et al. Generation of an immortalised erythroid cell line from haematopoietic stem cells of a haemoglobin E/β-thalassemia patient. Sci Rep. 2020;10:16798.

    Article  CAS  Google Scholar 

  8. Soboleva S, Kurita R, Ek F, Åkerstrand H, Silvério-Alves R, Olsson R, et al. Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines. Commun Biol. 2021;4:677.

    Article  CAS  Google Scholar 

  9. Tang S, Tao M, McCoy JP Jr, Zheng ZM. The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation. J Virol. 2006;80:4249–63.

    Article  CAS  Google Scholar 

  10. Liem RK. Cytoskeletal integrators: the spectrin superfamily. Cold Spring Harb Perspect Biol. 2016;8:a018259.

    Article  Google Scholar 

  11. Daniels DE, Downes DJ, Ferrer-Vicens I, Ferguson DCJ, Singleton BK, Wilson MC, et al. Comparing the two leading erythroid lines BEL-A and HUDEP-2. Haematologica. 2020;105:e389–94.

    Article  CAS  Google Scholar 

  12. Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science. 2008;322:1839–42.

    Article  CAS  Google Scholar 

  13. Sankaran VG, Xu J, Ragoczy T, Ippolito GC, Walkley CR, et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature. 2009;460:1093–7.

    Article  CAS  Google Scholar 

  14. Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol. 2005;33:259.

    Article  CAS  Google Scholar 

  15. Lee YT, de Vasconcellos JF, Yuan J, Byrnes C, Noh SJ, Meier ER, et al. LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo. Blood. 2013;122:1034–41.

    Article  CAS  Google Scholar 

  16. Miharada K, Hiroyama T, Sudo K, Nagasawa T, Nakamura Y. Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells. Nat Biotechnol. 2006;24:1255–6.

    Article  CAS  Google Scholar 

  17. Hiroyama T, Miharada K, Sudo K, Danjo I, Aoki N, Nakamura Y. Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells. PLoS ONE. 2008;3:e1544.

    Article  Google Scholar 

  18. Sankaran VG, Orkin SH, Walkley CR. Rb intrinsically promotes erythropoiesis by coupling cell cycle exit with mitochondrial biogenesis. Genes Dev. 2008;22:463–75.

    Article  CAS  Google Scholar 

  19. Sen T, Jain M, Gram M, Mattebo A, Soneji S, Walkley CR, et al. Enhancing mitochondrial function in vivo rescues MDS-like anemia induced by pRb deficiency. Exp Hematol. 2020;88:28–41.

    Article  CAS  Google Scholar 

  20. Le Goff S, Boussaid I, Floquet C, Raimbault A, Hatin I, Andrieu-Soler C, et al. p53 activation during ribosome biogenesis regulates normal erythroid differentiation. Blood. 2021;137:89–102.

    Article  Google Scholar 

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Acknowledgements

We thank Martin L Olsson, Johan Flygare, and Jenny Hansson for scientific discussion, and Yukio Nakamura and Alexander Doyle for critical reading. This work was supported by the Swedish Research Council (K.M.), the Swedish Cancer Society (K.M.), and Royal Physiographic Society of Lund (S.S.). K.M. was funded by StemTherapy program at Lund University. The Lund Stem Cell Center was supported by a Center of Excellence grant in life sciences from the Swedish Foundation for Strategic Research.

Funding

Vetenskapsrådet (The Swedish Research Council), Cancerfonden (The Swedish Cancer Society), Kungl. Fysiografiska Sällskapet i Lund (Royal Physiographic Society of Lund).

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Authors and Affiliations

  1. Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden

    Svetlana Soboleva & Kenichi Miharada

  2. Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan

    Ryo Kurita

  3. Division of Medical Microbiology, Lund University, Lund, Sweden

    Naoko Kajitani

  4. Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Lund, Sweden

    Hugo Åkerstrand

  5. International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan

    Kenichi Miharada

Authors
  1. Svetlana Soboleva
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  2. Ryo Kurita
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  3. Naoko Kajitani
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  4. Hugo Åkerstrand
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  5. Kenichi Miharada
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Contributions

KM designed the project. SS and KM planned experiments. SS performed experiments. RK provided materials. HÅ supported experiments. NK gave specialists opinions. SS and KM wrote the manuscript.

Corresponding author

Correspondence to Kenichi Miharada.

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Human bone marrow was donated from a healthy donor with a written informed consent.

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13577_2021_652_MOESM1_ESM.pdf

Supplementary file1 Supplementary Figure 1 (A) RT-PCR analysis of expression of HPV16-E6, HPV16-E7 and GAPDH genes in ELLU clones at steady state and on day 14 of differentiation. (B) Representative plots of flow cytometry analysis for the frequencies of Band3+CD49d+ and Band3+CD49d- cells in the differentiating ELLU clones (PDF 2734 kb)

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Soboleva, S., Kurita, R., Kajitani, N. et al. Establishment of an immortalized human erythroid cell line sustaining differentiation potential without inducible gene expression system. Human Cell 35, 408–417 (2022). https://doi.org/10.1007/s13577-021-00652-7

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  • DOI: https://doi.org/10.1007/s13577-021-00652-7

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