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Manufactured red blood cells
regenerative medicine product is anticipated to reduce transfusion frequency and the associated iron overload.6 Support for this idea comes from studies showing that transfusion of young RBC (also called neocytes) is benefi- cial to patients with inherited anemias, reducing iron over- load and increasing the interval between transfusions.8-12
mRBC have been tested in immunocompromised mice models5,6,13 and non-human primates.13 Importantly, a proof-of-principle mini-transfusion of autologous mRBC has been conducted in a single volunteer, illustrating that mRBC can survive in the circulation and are safe for use in humans.5 The benefit of mRBC in allogeneic transfusions across multiple recipients still needs to be demonstrated before moving to patients. The commercial company Rubius Therapeutics has a business model built around producing novel mRBC for therapeutics. To date, Rubius has conducted one injection of mRBC engineered for treatment of phenylketonuria in a single patient (Rubius press release 12th March 202014), but no further specific information was released. In the UK, the National Health Service Blood and Transplant (England’s Blood Service) is intending to conduct a single-center, randomized, allo- geneic, controlled, phase I, cross-over trial denominated RESTORE (Recovery and Survival of Stem Cell Originated Red Cells (ISRCTN:42886452 and EudraCT: 2017-002178- 38). This healthy volunteer trial has faced significant delays, most recently due to the COVID-19 pandemic but will, it is hoped, be carried out in the near future to assess the recovery and survival of a mini-dose of mRBC derived from CD34+ cells isolated from adult blood donors versus the standard RBC from the same donor.
Laboratory-grown RBC offer the greatest potential in terms of sourcing rare blood groups for sickle cell and tha- lassemia patients with alloimmunity. It must be acknowl- edged however, that these patients also present the great- est challenge in terms of requirements for blood. Adult patients require multiple units of blood per month.15 Realistically, the first therapeutic use of mRBC is likely to take place in a pediatric setting or for red cell-based thera- peutics, such as enzyme replacement therapies, as both these applications require smaller numbers of mRBC. There is also a need to determine the number of mRBC that represents a therapeutic dose. For adult patients, one unit of standard RBC is estimated to consist of approxi- mately 2x1012 RBC which raises the hemoglobin of an average adult by 1 g/dL. For pediatric patients, doses are more variable as they depend on the weight of the patient but are lower than an adult dose. It should be noted that a proportion (5-10%) of standard RBC are lost within the first 24 hours after transfusion16 and this increases to 25% or more with blood storage time. Therefore, the actual number of RBC required to treat anemia is likely to be lower if the majority of the cells are nascent.
Many excellent reviews have been written on mRBC and the prospect of using mRBC for transfusion.17-25 We therefore offer below a concise overview of the progress to date, highlighting the relevant issues and opportunities for optimizing and increasing the mRBC yield to an adult therapeutic dose.
Overview of the erythroid culture process
The recapitulation of erythropoiesis using primary hematopoietic stem and progenitor cells (HSPC) ex vivo
requires specific combinations of cytokines and growth factors in order to first expand the HSPC, and then to direct lineage specification to ensure full differentiation to the reticulocyte stage (see Figure 1). Over the last 20 years, multiple laboratories have developed two-dimensional liquid culture systems that reproduce the process and stages of human erythropoiesis to generate reticulocytes. These include two to four stages, each characterized by the inclusion or omission of specific growth factors (Table 1). The general consensus is for the inclusion of a primary stage favoring HSPC expansion with interleukin-3 and stem cell factor, a secondary erythroblast expansion stage including stem cell factor and erythropoietin, followed by a terminal differentiation stage with erythropoietin. Notably, holotransferrin is included throughout the cul- ture period. Some laboratories further modify the initial- stage culture media by including, for example, throm- bopoietin and fms-like tyrosine kinase 3 (Flt-3) to enhance stem cell proliferation13 and may also include glucocorti- coids to increase expansion prior to differentiation.26 The more recent culture protocols listed in Table 1 have been undertaken at considerably larger scale (i.e., at least 1 L), with some reports of successful generation of large num- bers of reticulocytes. The challenge for the field is to increase the production even further to generate the equivalent of a therapeutically useful adult dose.
Starting material
The studies reporting the highest yields all use HSPC specifically isolated from cord blood,13,27 mobilized5 or standard peripheral blood6,28 (Table 1). Another option is to use the whole peripheral blood mononuclear cell (PBMNC) component for production of mRBC, thereby omitting the expensive step of CD34+ isolation.7,29 As well as reducing costs, the use of PBMNC as the starting mate- rial under the correct culture conditions can contribute towards increasing the yield of mRBC. Indeed, PBMNC include all cells with erythroid lineage potential, some of which are CD34- that can enhance culture yield.29 PBMNC also include CD14+ cells that might act as helper/feeder cells that can limit the cell death of erythroid progenitors during the first few days of culture when volumes are still small and cells are kept in static tissue culture flasks or dishes.30,31
We highlight that there are other sources that are gain- ing traction, including immortalized pluripotent stem cells and immortalized cell lines,32,33 which can be used to dif- ferentiate to reticulocytes, but we will not discuss these here because these have not yet been grown at scale. These sustainable cellular sources have great potential for continual blood production once the technical challenges of growing them have been circumvented and are likely to comprise a second wave of blood products after stem cell- derived mRBC.
Natural donor variation and yields
The genetic makeup of the donor-derived cellular start- ing material has long been recognized to have an impact on yield, which is problematic when trying to consistently produce a high number of mRBC using random donors.34 This variation could be due to the number of HSPC pres-
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