Abstract
Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical quality blood, particularly for individuals with rare blood group phenotypes. Immortalised erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show our immortalisation approach is reproducible for erythroid cells differentiated from bone marrow, and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterisation of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalisation. In addition, we show only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalisation. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.
| Original language | English |
|---|---|
| Pages (from-to) | 26-39 |
| Number of pages | 14 |
| Journal | Molecular Therapy - Methods and Clinical Development |
| Volume | 22 |
| Early online date | 11 Jun 2021 |
| DOIs | |
| Publication status | Published - 10 Sept 2021 |
Bibliographical note
Funding Information:The study was supported by NHS Blood and Transplant (NHSBT) and the NIHR Blood and Transplant Research Unit (NIHR BTRU) in Red Cell Products (IS-BTU-1214-10032). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care. The study was also supported by MRC grant MR/S021140/1. The authors would like to thank Dr. Kate Heesom, Director of the Bristol University Proteomic Facility, UK for performing mass spectrometry, the University of Bristol Flow Cytometry Facility for use of equipment, and the Wellcome Trust Clinical Research Facility at the University of Edinburgh, UK for performing molecular karyotyping. IBGRL antibodies were kindly supplied by Mr. Jonathan Dixey, IBGRL, Filton, Bristol, UK. The IGF2BP1 antibody was a gift from Professor Stefan H?ttelmaier, Martin Luther University, Germany. J.F. conceived and supervised study. Experiments were conceived and designed by J.F. and D.E.D. with contribution from K.T. The BEL-P cell line was generated by D.E.D. and K.T. BEL-C by D.E.D. and BEL-A td3, 5, 7, and 9 lines by R.E.G. and N.C. with input from J.F. K.T. and D.J.A. D.E.D. and D.C.J.F. conducted the majority of experiments, analyzed data, and prepared figures. R.E.G. K.T. N.C. K.A.M. T.A. K.E.M. I.F.-V. and D.R.J. conducted experiments. P.A.L. performed proteomics analysis. M.C.W. carried out the HPLC experiments and contributed to proteomics analysis. K.A.M. contributed to proteomics analysis. M.A.C. analyzed molecular karyotyping data. R.K. and Y.N. provided the HPV16 E6 and E7 construct. D.E.D. and J.F. wrote the manuscript. D.J.A. edited the manuscript. The authors declare no competing interests.
Funding Information:
The study was supported by NHS Blood and Transplant (NHSBT) and the NIHR Blood and Transplant Research Unit (NIHR BTRU) in Red Cell Products ( IS-BTU-1214-10032 ). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care. The study was also supported by MRC grant MR/S021140/1 . The authors would like to thank Dr. Kate Heesom, Director of the Bristol University Proteomic Facility, UK for performing mass spectrometry, the University of Bristol Flow Cytometry Facility for use of equipment, and the Wellcome Trust Clinical Research Facility at the University of Edinburgh, UK for performing molecular karyotyping. IBGRL antibodies were kindly supplied by Mr. Jonathan Dixey, IBGRL, Filton, Bristol, UK. The IGF2BP1 antibody was a gift from Professor Stefan Hüttelmaier, Martin Luther University, Germany.
Publisher Copyright:
© 2021