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May-Grünwald Giemsa staining of in vitro produced red blood cells throughout the
different stages of the erythroid differentiation process. Scale bar: 10 µm.

What?

Cultured red blood cells (cRBCs) are cells grown in vitro in the laboratory using different hematopoietic stem cell (HSC) sources recapitulating erythropoiesis in vivo. This is a complex process in which HSC proliferate and give rise to erythroid committed progenitors which in turn differentiate to enucleated red cells. Erythroid precursors are defined morphologically as proerythroblasts (ProE), basophilic erythroblasts (Baso), polychromatic erythroblasts (PolyE), and orthochromatic erythroblasts (OrthoE). OrthoE enucleate to form reticulocyte (Retic) which finally become mature red blood cells in vivo (1).

Why?

Blood transfusion is an indispensable procedure in modern healthcare systems, where transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Blood supply is currently dependent mainly on volunteer donations. However, the imbalance between blood demand and supply is increasing due to population ageing. In developed countries, the need for blood is predicted to increase while the number of healthy blood donors will decline. Moreover, the limited availability of compatible red cell units for patients with rare blood types or with multiple alloantibodies is a major challenge for the current blood supply system. Thus, the in vitro production of cultured and customizable red cells has been widely investigated in an attempt to alleviate the dependence on blood donation and resolve unmet clinical needs.

Who?

The main potential applications for in vitro produced red cells are: 1) Transfusion support of immunized patients with rare blood group phenotypes; 2) Provision of phenotype-compatible RBC units for specific groups of patients requiring chronic transfusions (particularly, for patients of African origin suffering red cell disorders such as sickle cell disease who require regular transfusions); and 3) Production of reagent-red cells for their use in diagnostic assays. Cultured red cells have already been used in the laboratory for experimental purposes; however, the clinical use of such a product is not yet a reality. Of relevance, the first clinical trial in humans using in vitro grown RBCs from peripheral blood CD34+ cells (RESTORE) is presently underway in the UK: RESTORE - Clinical Trials Unit - NHS Blood and Transplant (nhsbt.nhs.uk). 

When?

Up to now, several strategies have been considered in an attempt to generate RBCs in vitro. The initial research started with human adult HSCs from cord blood, bone marrow and mobilized peripheral blood stem cells (2, 3). Despite the good results obtained with these starting materials, primary adult stem cells have limited proliferation potential in vitro. Since 2006, when Yamanaka and Takahashi confirmed the concept of cellular plasticity, this led to the hiPSCs (human induced pluripotent stem cells) technology, which opened the door to generating patient-specific cells for cell therapy (4). However, erythrocytes derived from hiPSCs present a fetal phenotype and the whole differentiation process is inefficient since the enucleation rate is in the range of 1-2%. In 2013, Kurita et al. established immortalized erythroid progenitor cell lines by transformation with the HPV16-E6/E7 proteins (5). In 2017, Trakarnsanga et al. established an immortalized erythroid cell line derived from adult bone marrow CD34+ cells through transduction with the Tet-inducible HPV16-E6/E7 expression system. This cell line named BEL-A2 (Bristol Erythroid Line Adult) could proliferate for more than 5 months and has the ability to mature into functional reticulocytes with an adult phenotype (6). Thereafter, other groups have described the generation of other immortalized erythroid cell lines (imERYPCs) (7, 8). These cells are capable of providing an unlimited supply of red blood cells with an adult phenotype in addition to avoiding the complex and lengthy differentiation process required by hiPSCs. The continuous efforts to establish advanced strategies for a cost-effective, highly potent RBC culture system combined with engineering techniques, have contributed towards the in vitro generation of RBCs for future clinical use.

Where?

The Immunohematology Research Group of the Banc de Sang i Teixits (BST) in Barcelona, led by Dr. Núria Nogués, has worked on the topic of in vitro produced red blood cells over the past 6 years in different funded collaborative projects. First, focusing on the establishment of hiPSC lines from selected blood donors and more recently, moving to the strategy of immortalizing erythroid progenitor cells. Currently, we are working in two projects. One of them, funded by ‘la Caixa’ foundation, aims to develop a novel bioprocess to scale-up red cell production from immortalized erythroid precursor lines with infrequent blood groups required for the safe transfusion of sickle cell disease patients. This project is in collaboration with the Prof. Jan Frayne’s Research Group at the University of Bristol. In parallel, the project entitled “Lab-grown red cells from immortalized erythroid precursor cell lines as an alternative source of red cells with rare phenotypes for diagnostic and therapeutic applications” is funded by ‘El Ministerio de Ciencia e Innovación’ and is part of the program for Knowledge Generation.

How?

The methodology to generate in vitro red blood cells is based on the approach of immortalizing early erythroblasts from adult peripheral blood CD34+ cells. Firstly, peripheral blood Mononuclear Cells (PBMNCs) are collected from selected blood donors. The PB CD34+ cells are isolated by immunomagnetic positive selection and cultured in the erythroid differentiation medium for 24h.Then PB CD34+ cells are transduced with a lentiviral vector using a Tet-inducible HPV16 E6/E7 system (6, 9) which allows immortalisation of cells at a specific time-point during erythroid differentiation. Finally, proliferating imERYPC lines are transferred to differentiation culture media for up to 12 days with specific cytokines to obtain the final cultured RBC.

Did you know that …

One blood transfusion unit contains approximately 2 x 1012 red blood cells, which would require more than 1000 L of medium to produce that many cells in static flask cultures. To date, there is no production system capable of sustaining the large-scale production required for clinical use. In our laboratory, the use of a bioreactor system is being optimized for an efficient and scalable production of cultured red blood cells and, in this way, address the challenge of up scaling the cultures of imERYPCs to the required volume for their potential use.

References

1. Palis J. Primitive and definitive erythropoiesis in mammals. Vol. 5 JAN, Frontiers in Physiology. 2014.
2. Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol. 2005 Jan 1;23(1):69–74.
3. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. Haematologica. 2017 Feb 28;102(3):476–83.
4. Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell. 2006 Aug 25;126(4):663–76.
5. Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, et al. Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells. PLoS One. 2013 Mar 22;8(3).
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 Mar 14;8. 
7. Bagchi A, Nath A, Thamodaran V, Ijee S, Palani D, Rajendiran V, et al. Direct generation of immortalized erythroid progenitor cell lines from peripheral blood mononuclear cells. Cells. 2021 Mar 1;10(3):1–18.
8. Scully EJ, Shabani E, Rangel GW, Grüring C, Kanjee U, Clark MA, et al. Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion. Am J Hematol. 2019 Sep 1;94(9):963–74. 
9. 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 Dec 1;10(1).