Generation of immortalized adult erythroid line

We set out to create immortalized adult erythroid lines, utilizing a Tet-inducible HPV16-E6/E7 expression system8. Adult bone marrow CD34+ cells were transduced with the HPV16-E6/E7 construct and maintained in primary medium of our erythroid culture system3 for 4 days. On day 5 cells were transferred to expansion medium containing doxycycline to induce expression of E6 and E7, and were maintained in this medium thereafter. Figure 1a shows a schematic of the protocol. The cells proliferated continuously and after 190 days, well beyond the Hayflick limit11, were frozen for storage. Samples were also frozen at regular intervals throughout this time period. All samples re-established efficiently in culture following freeze thawing. The line was named BEL-A for Bristol Erythroid Line Adult. The mean doubling time of the cells after day 100 in continuous culture was 20 h. Morphological analysis of the immortalized cells showed that they are pro- to early basophilic erythroblasts. There was no change in morphology over time (Fig. 1b).

Figure 1: Generation of immortalized adult erythroid cell line. Human adult bone marrow CD34+ cells were recovered from frozen primary erythroid culture medium for 24 h, before transduction with Tet-inducible HPV16 E6/E7 construct. Cells were transferred to StemSpan expansion medium with doxycycline on day 5 in culture and maintained in expansion medium thereafter. (a) Schematic of experimental approach. (b) Representative cytospins illustrating similar morphology of cells maintained proliferating in continuous culture on days 57, 125 and 164. (c) Representative cytospin images showing morphology of cells on days 4, 10 and 18 following transfer to erythroid differentiation medium and of reticulocytes isolated by filtration. Cytospins of erythroid cells differentiated from adult peripheral blood CD34+ at comparative stages of differentiation are included for comparison. Cells were stained with May–Grunwald Giemsa reagent and analysed by light microscopy. Scale bars 10 μm. Full size image

BEL-A cells after 100 days in continuous culture were transferred to primary erythroid culture medium containing doxycycline for 6 days, and then to tertiary medium3 without doxycycline to induce differentiation to mature erythroblasts (orthochromatic normoblasts) and reticulocytes (Fig. 1c). Comparison with erythroid cells differentiated from adult PB CD34+ progenitor cells showed similar morphological progression down the erythroid pathway (Fig. 1c). BEL-A cells transferred to differentiation medium at regular time points post day 100 in culture showed unchanged ability to differentiate. The expansion profile of such cells was also consistent (Supplementary Fig. 1). Cells enucleated up to 30%, with stable reticulocytes isolated by filtration using a standard leukocyte reduction filter3 (Fig. 1c). Cultured adult reticulocytes were 9.8±1 μm (mean±s.d., n=100) in diameter and BEL-A reticulocytes 9.9±1 μm (mean±s.d., n=100). We confirmed that expression of HPV16 E6 and E7 was lost from BEL-A cells following removal of doxycycline (Supplementary Fig. 2).

Characterization of the BEL-A line

Morphologically, erythroid cells and reticulocytes differentiated from the BEL-A line appear identical to those derived from adult PB CD34+ cultures. We next extensively characterized the properties of BEL-A cells in comparison with these adult erythroid cells. Extensive characterization of in vitro adult reticulocytes has been carried out previously and reveals no substantive differences compared to endogenous cells3,12,13. Furthermore, the cultured reticulocytes survive and mature in vivo12,14,15.

Erythroid cells undergo distinct changes in their membrane protein expression profile during erythropoiesis, with some proteins increasing and others decreasing in level as the cells differentiate16,17,18. To determine if BEL-A cells exhibit a normal expression profile of membrane proteins during differentiation we compared protein levels during erythropoiesis with that of erythroid cells differentiated from adult PB CD34+ cells by flow cytometry, using antibodies to a panel of established RBC antigens. The dynamic expression pattern was equivalent for both populations of cells (Supplementary Fig. 3a,b). We also compared the abundance of glycophorin A (GPA) and band 3 in BEL-A reticulocytes with that of in vitro adult reticulocytes and endogenous RBCs (Supplementary Fig. 3c). The level of both proteins was equivalent in the reticulocyte populations, but lower in RBCs, as expected. The data also confirmed that the reticulocytes are larger than RBCs. Reticulocytes loose volume and surface area via exosomes and vesicles3,19,20, particularly as they mature in the circulation. GPA and band 3 have been shown to be present on such vesicles3,19, thus reducing abundance on the mature RBC.

Serological analysis confirmed that the blood group of the BEL-A reticulocytes corresponds to that of the bone marrow CD34+ cell donor from which they were created. Importantly, the BEL-A reticulocytes do not have T or Tn antigen exposure demonstrating normal O-glycosylation.

Before differentiation centrifuged BEL-A cells produced a pink pellet, but a red pellet following differentiation, indicating haemoglobin synthesis. We compared the globin expression profile of BEL-A cells to that of erythroid cells differentiated from adult PB CD34+ at the same stage of differentiation. Erythroid cells differentiated from CB CD34+ cells were included as a positive control for γ-globin expression, as well as cells from one of the previously generated immortalized erythroid lines, HiDEP-1 (ref. 8). BEL-A cells synthesized exclusively β-globin at the level detected in adult erythroid cells; densitometry of the globin bands on western blot gave a ratio of BELA:PB β-globin of 1.04 (Fig. 2a). In contrast, HiDEP-1 cells expressed predominantly ɛ- with some γ-globin, as we have shown previously21. These data were corroborated by HPLC globin typing whereby erythroid cells at day 20 from an adult culture contained 94.9% HbA, 3.1% HbA2 and 2% HbF, BEL-A cells at day 12 in culture contained 99.3% HbA and 0.7% HbF and HiDEP-1 cells contained 3.8% HbA, 40.3% HbF and 55.9% HbE. We compared the level of transcription factors KLF1 and BCL11A in these cells since both are required for the switch to and expression of β-globin21. The level of KLF1 transcripts in BEL-A and HiDEP-1 cells was similar to that detected in adult erythroid cells. However, whereas BEL-A cells expressed normal adult levels of BCL11A, no transcripts were detected in HiDEP-1 cells, consistent with their failure to express β-globin (Fig. 2b). A number of other key erythroid transcription factors in BEL-A cells were expressed equivalently to adult erythroid cells at a similar stage of differentiation (Supplementary Fig. 4).

Figure 2: Characterization of BEL-A line. (a) Western blots of late stage BEL-A, HiDEP-1 and erythroid cells differentiated from adult PB and cord blood CD34+ cells (at days 18, 12, 19 and 18 in culture, respectively) incubated with antibodies to α-, β-, γ- and ɛ-globin. (b) Expression of BCL11A and KLF1 transcripts in expanding BEL-A and HiDEP-1 cells and erythroid cells differentiated from adult PB CD34+ cells at days 5 and 8 in culture by RT-PCR. Transcripts for AHSP were included as a positive control. (c) Erythroid cells differentiated from adult peripheral blood CD34+ cells (i) and BEL-A cells (ii) were fixed, permeabilized and dual stained for F-actin (red) and myosin IIb (green). Single cells showing F-actin and myosin IIb localization in an enucleating cell, and in a reticulocyte are shown in three dimension. (d) Filtered adult reticulocytes (i) and BEL-A reticulocytes (ii) were live imaged after staining for phosphatidylserine (PS), glycophorin A intracellular domain (BRIC163) and band 3 intracellular domain (BRIC155) (all green). Scale bars 5 μm. Full size image

A major barrier to the use of PSCs for the manufacture of red cells is the failure of erythroblasts generated to enucleate correctly. F-actin and myosin are known to form a contractile actin ring proximal to the site of nuclear extrusion22,23, with myosin IIb implicated in enucleation24. We show that Myosin IIb is a component of the F-actin core, with co-localization apparent at the point of nuclear extrusion and persisting in early reticulocytes generated from PB derived CD34+ cells (Fig. 2c). This apparent association between F-actin and myosin IIb is also seen in BEL-A cells (Fig. 2c). In contrast, in HiDEP-1 cells, which are derived from induced PSCs, enucleated cells are fragile and morphologically abnormal with an irregular plasma membrane and lack a myosin and F-actin core (Supplementary Fig. 5).

The vesicles extruded from reticulocytes during maturation to an erythrocyte have been shown to be large, intact, inside-out phosphatidylserine-exposed autophagic vesicles19. Live cell imaging of filtered BEL-A reticulocytes confirmed the presence of such vesicles at the cell surface (Fig. 2d), further demonstrating their comparability with adult cultured reticulocytes.

Comparing the proteome of BEL-A and adult reticulocytes

Comparative proteomics of BEL-A reticulocytes (three replicates) and reticulocytes isolated from cultures of PB CD34+ cells from three separate individuals were performed. No proteins detected were unique to the BEL-A or in vitro-cultured adult reticulocytes. Of the quantified proteins, 1964 were common to all BEL-A and adult replicates (Supplementary Data 1). Of these, 19 (<1%) were more abundant in the BEL-A reticulocytes and 191 (9.7%) in the adult reticulocytes (Supplementary Data 2a,b). Notably, 31% of the latter were ribosome proteins and 15% mitochondrial proteins, known to be progressively lost during reticulocyte maturation (reviewed in Ney (2011)20). Upon further analysis, and cross reference with our data mapping the change in proteome between in vitro-cultured adult reticulocytes and RBCs, it was apparent that the differences were due to the stage of reticulocyte maturation, with a shift in the relative distribution between the populations: heterogeniety in maturation of reticulocyte populations having previously been reported25. The data files were also searched against the Uniprot human papillomavirus type 16 database, with no viral proteins detected in the reticulocyte samples.

Functional analysis of BEL-A cells

We also addressed BEL-A red cell function. Normal deformability of the BEL-A reticulocytes is essential for clinical use. Comparison of BEL-A and normal adult reticulocytes using an Automated Rheoscope Cell Analyser26 showed comparable deformability indexes (Supplementary Fig. 6). The cells also bound and released oxygen in a manner comparable to normal adult erythroid cells (Supplementary Fig. 7).

In vivo survival of BEL-A reticulocytes

To evaluate survival and maturation of BEL-A reticulocytes in vivo, macrophage-depleted NSG mice15 were transfused with BEL-A cells or adult donor RBCs. Overall, there were no difference in the survival rate of BEL-A and donor RBC detected in the murine circulation over the evaluation period (Supplementary Fig. 8a,b). The diameter of BEL-A cells decreased between 10 min and 24 h post transfusion (Supplementary Fig. 8c), with cells at 24 h exhibiting mature erythrocyte morphology (Supplementary Fig. 8d).

Large-scale culture of BEL-A cells

To evaluate the feasibility of transfer to larger scale production, BEL-A cells grown under static culture conditions to 250 ml were transferred to 1.5 l spinner flasks (Supplementary Fig. 9). The doubling rate of the cells was unchanged and they differentiated normally on transfer to differentiation medium, demonstrating that the cells will grow in stirred vessels as well as in static culture, which is important for development of future scale-up procedures and demonstrates a potential pathway to therapeutic productivity. To date, optimal protocols for such large-scale culture and bioreactor vessels for the manufacture of red cells at a clinical scale have not been reported, but the same hurdles will apply for erythroid cultures using all stem cell sources as progenitors. The feasibility of developing such a system, along with associated projected costs, has been extensively reviewed27,28,29. Alongside further work required to optimize enucleation, reticulocyte stability in culture and efficient methods for reticulocyte purification required for escalation of culture yields, one of the next steps for our procedure is transfer to GMP conditions. We do not envisage significant hurdles with such transfer, as, for example Timmins et al.30 using a similar erythroid differentiation system found no effect on culture performance when fully animal component-free media was substituted.

Amenability of BEL-A cells to genetic manipulation

The BEL-A line is a valuable research resource, superior to any presently available for the study of erythropoiesis and red blood cells disorders. To illustrate the utility of these cells for genetic manipulation BEL-A cells were transduced with GPA-GFP, band 3-GFP, Lifeact-GFP and RhAG shRNA. Transduction efficiency of the GFP constructs using lentivirus was extremely high (average 98%; Fig. 3a), and the tagged proteins correctly localized to the plasma membrane. Knock down of RhAG with shRNA achieved a striking reduction in the level of protein (average 96%; Fig. 3b), and this reduction was maintained during expansion of the cells, throughout differentiation and in the resultant reticulocytes, demonstrating the capacity for generating sublines that mimic rare phenotypes. Expression of the transduced genes and RhAG was maintained at the same level following freeze thawing of the cells.