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S. Pellegrin et al.
ent per volume of blood, which is highly variable between donors, or down to subtle variations in genetic factors that influence how HSPC and erythroid progenitors prolifer- ate, enucleate and/or respond to culture media composi- tion. More work is therefore needed to explain the differ- ences in yields observed between donors under standard- ized and reproducible culture conditions. The payoff from this painstaking work would be the identification of potential genetic variations that could be pre-screened for or utilized by genetically engineering alterations the start- ing material to benefit production.
Genome-wide association studies as well as identifica- tion of rare phenotypes linked to specific RBC traits might help to identify genetic variants suitable for reliably pro- ducing large numbers of mRBC. An example of such an approach was carried out by Sankaran’s group.35 LNK/SH2B3 is an adaptor protein that negatively regulates hematopoietic cytokine signaling. Rare lost-of-function SH2B3 alleles have been associated with JAK2-mutation- negative erythrocytosis36,37 and a hypomorphic allele of SH2B3 (single nucleotide polymorphism rs3184504) was found to be significantly associated with high hemoglobin levels, packed cell volume and RBC count in vivo.38 Using shRNA knockdown in adult, mobilized, peripheral blood and cord blood CD34+ cells, Giani et al.35 suppressed the expression of the LNK/SH2B3 protein and reported a 2- to 7-fold increase in yield of enucleated RBC in shRNA-treat- ed cells compared to cells transduced with a control shRNA. More recently, a study of rare MAM-negative individuals by Thornton and colleagues39 showed that
peripheral blood CD34+ cells from two MAM-negative individuals had a proliferation advantage in ex-vivo ery- throid cultures, resulting in an average 5-fold increase in cell number compared to four age- and gender-matched MAM-positive controls. Whether the same observations concerning loss-of-function SH2B3 and MAM-negative cells hold true for large-scale cultures and across multiple donors still needs to be determined.
Beyond yield, the choice of donor can also affect the quality of the final mRBC product due to the donor’s own RBC intrinsic characteristics – not just in terms of blood group which can be selected for, but also in terms of stor- age characteristics or even longevity in circulation once transfused. The planned RESTORE clinical trial may pro- vide data on this as the survival time of transfused stem cell-derived mRBC in the circulation of the recipient will be directly compared to the survival time in circulation of the same donor’s standard RBC.
Genetic manipulation and small molecules
A key challenge for the field is to prevent the attrition of the self-renewal capacity of HSPC and to maintain the expansion capacity of erythroid progenitors (burst-form- ing and colony-forming units-erythroid) for a longer peri- od before terminal erythroid differentiation occurs. One way to do this is through the use of glucocorticoids (see below) which can potentially improve the asynchronicity of enucleation in cultures, and may then improve reticulo-
Figure 1. Overview of the erythroid culture process. Ex vivo culture systems require the isolation of peripheral blood mononuclear cells (PBMNC) or magnetic sorting of the CD34+ cells as starting material. Culture systems then employ either flasks, spinner flasks or a bioreactor system depending on scale. The volume of the culture will increase dramatically as the cells expand and differentiate through days 7-14. Upon generation of a mixed population of reticulocytes, nucleated cells and pyreno- cytes at day 21, cells then require filtration using either a syringe (small scale) or multiple leukocyte filters (large scale) depending on volume to give a pure reticulo- cyte population. Diagram made using biorender.com.
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haematologica | 2021; 106(9)