Skip to main content

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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  Google Scholar 

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

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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.

    CAS  Article  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 

Download references

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).

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

Conflict of interest

There is no conflict of interest.

Ethical approval

Not applicable.

Informed consent

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

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

Keywords

  • Red blood cells
  • Immortalized erythroid cell lines
  • HPV-E6/E7
  • Enucleation
  • Hemoglobin