0.00

50000.00

100000.00

150000.00

200000.00

250000.00

300000.00

350000.00

400000.00

d0 d2 d4 d6 d8 d10 d12 d14 d16 d18

Supplementary Figure 1. Expansion profile of BEL-A cells following transfer to differentiation

medium. Data shown are mean±s.d. n=3.

BEL

-A d

0

BEL

-A d

5

BEL

-A d

9

BEL

-A d

15

PB

d5

PB

d8

PB

d1

1

PB

d1

5

wat

er

HPV E7

GAPDH

HPV E6

Supplementary Figure 2. Expression of HPV16 E6 and E7

BEL-A cells were maintained in expansion medium with doxycycline (day 0) before transfer to primary

differentiation medium with doxycycline (day 1- 6) and tertiary differentiation medium (day 7 onwards).

Erythroblasts differentiated from adult peripheral blood CD34+ cells were collected at day 5, 8, 11 and 15 in culture.

Expression of HPV16 E6 and E7 was determined by the PCR. Expression of GAPDH was included as a positive

control. The PCR reaction in lane ‘water’ did not contain cDNA.

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

GPA

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

GPC

CD147

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

CD44

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

α4 integrin β1 integrin

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

Band 3

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

CD71

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

Rh

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

RhAG

0

20

40

60

80

100

120

PB d5/ undif.BELA

PB d12/BELA d4

PBd21/BELAd10

Kell

PB

BEL-A

Supplementary Fig. 3a

GPA Kell GPC Control

3.00% 83.16% 36.98% 98.86%

3.29% 99.68% 98.75% 99.83%

3.14% 99.38% 81.79% 97.86%

CD147

99.97%

99.81%

99.82%

CD71

99.80%

99.64%

72.82%

Undif.

Dif. d4

Dif. d 10

Rh RhAg Band3

48.95% 95.43% 5.71%

98.90% 99.51% 98.70%

94.39% 97.68% 97.34%

CD44

98.84%

99.46%

88.15%

α4 β1

98.61% 99.13%

99.68% 99.83%

14.03% 18.42%

Undif.

Dif. d4

Dif. d10

Supplementary Fig. 3b

IgG

Adult cultured reticulocytes

BEL-A derived reticulocytes

GPA (BRIC256)

FSC

RBC Adult cultured reticulocytes

BEL-A derived reticulocytes

band 3 (BRIC71) GPA(BRIC256) FSC

Control RBC

Supplementary Fig. 3c

Supplementary Figure 3. Level of RBC membrane proteins in BEL-A compared to normal

adult erythroid cells during erythropoiesis

BEL-A cells in expansion medium (undif), and at day 4 and 10 following transfer to differentiation

media, and erythroblasts differentiated from adult peripheral blood CD34+ cells at day 5, 12 and

21 in culture were incubated with antibodies to the proteins indicated and analysed by Flow

Cytometry. (a) BEL-A cells compared to adult erythroid cells; y-axis indicates number of cell

displaying fluorescent intensity (b) Histograms illustrating fluorescent intensity of BEL-A cells

incubated with antibodies to indicated proteins at labelled stages of differentiation. Percentages

refer to percentage positivity (c) Endogenous RBCs, BEL-A reticulocytes and in vitro adult

reticulocytes were incubated with antibodies to glycophorin A (GPA) and band 3 (top row).

Forward scatter (FSC) is also shown as an indicator of cell size. GPA expression again FSC is

also shown (bottom row).

BEL

-A

PB

day

5

PB

day

8

wat

er

BCL11A

KLF1

AHSP

KLF3

GATA1

FOG1

SOX6

E2F2

NFE2

Supplementary Figure 4. Expression of key erythroid

transcription factors in BEL-A compared to normal

adult erythroid cells

RNA was isolated from BEL-A cells maintained in

expansion medium and erythroblasts differentiated from

adult peripheral blood CD34+ cells at day 5 and 8 in culture.

Expression of transcription factors was analysed by PCR.

Expanding BEL-A cells are at a stage of differentiation

closer to that of adult cells at day 5 than day 8 in culture.

HiDEP-1

cells

F-actin Myosin IIb

F-actin

Supplementary Figure 5. Re-localisation of F-actin and myosin at enucleation in HiDEP-1 cells.

Differentiating HiDEP-1 cells were fixed, permeabilsed and stained for F-actin and myosin IIb (red).

Enucleating HiDEP-1 cells show cytoskeletal protein mis-localisation (upper panel). HiDEP-1

reticulocytes (white arrows) are morphologically abnormal (lower panel). Scale bars are 5um.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8

RBC

ADULT RETICS

BELA2 RETICS

ADULT RETICS

RBC

BEL-A RETICS

Elongation index (A/B)

f

Supplementary Figure 6. Deformability of BEL-A reticulocytes

Adult reticulocytes (3-5 x106) differentiated from adult PB CD34+ cells, BEL-A derived reticulocytes or donor red blood

cells resuspended in 200µl PVP solution (viscosity at 37C 28.1cP) were subjected to shear stress of 3Pa using an

Automated Rheoscope Cell Analyser. At least 400 images per sample were analysed and the distribution of elongation

indexes plotted in Microsoft Excel.

Supplementary Figure 7. Oxygen binding of BEL-A erythroid cells

The oxygen dissociation curve was plotted for 20μl of pelleted BEL-A cells (differentiation day 13), cultured adult erythroblasts

(day 14, 16% reticulocytes) or adult human peripheral blood

BEL-A p50 25.59

Donor RBC p50 25.79

Adult culture p50 29.66

Oxygen dissociation

% o

xyh

emo

glo

bin

P O2

6.15

6.07

5.77

5.41

5.65

6.62

5.91

5.84

5.72

0.53

0

2

4

6

8

10

12

10m 8h 24h

Ce

ll D

iam

ete

r μm

Time post transfusion

c

a b

Supplementary Fig. 8

10min 24hr

3D 3D

d

Supplementary Figure 8. Survival and maturation of BEL-A cells in macrophage depleted NSG mouse

BEL-A or adult donor RBCs were inoculated into the lateral tail vein of macrophage depleted NSG mice.

Peripheral blood aspirates were taken from the opposite lateral tail vein at the time points indicated. Human cells

were detected by expression of glycophorin A using flow cytometry (a) Percentage of human donor RBC or BEL-

A cells in the mouse circulation (b) Proportion of human donor cells or BEL-A cells, normalized to 100% at 10

minutes after injection. Data represent mean±s.e.m. n=3 (c) The diameter of BEL-A cells measured at 10

minutes, 8 and 24 hours post transfusion Data represent mean±s.d. n=12 (d) 3D representation of BEL-A cells at

10 minutes and 24 hours post transfusion. Scale bars 5µm

Supplementary Figure 9. Scalability of the BEL-A line

BEL-A cells grown in static culture to 250ml were transferred to 1.5L spinner flasks. Expansion and differentiation

was consistent to that of cells in static culture.

BEL-A cells in spinner flasks

17 25 30 46

17 25 30 46

17 25 30 46

17 25 30 46

BEL

-A1

PB

CB

HiD

EP-1

α-globin β-globin

γ-globin ε-globin

BEL

-A2

BEL

-A 1

PB

CB

HiD

EP-1

BEL

-A 2

BEL

-A 1

PB

CB

HiD

EP-1

BEL

-A 2

BEL

-A 1

PB

CB

HiD

EP-1

BEL

-A 2

BEL

-A 1

PB

CB

HiD

EP-1

BEL

-A 2

Supplementary Figure 10. Original film with full blots from which portions in boxed areas are shown in Figure 2a. The

BEL-A cells described in the manuscript are in lane 2 of each blot (BEL-A 2). Globin expression in one of our additional

immortalised adult erythroid lines is shown in lane 1 of each blot (BEL-A 1)

70 kD

50 kD

37 kD

25 kD

100 kD

150 kD

250 kD

RhAG

GAPDH

Supplementary Figure 11. Full blot from which portion in boxed area is shown in Figure 3.

Membrane was also incubated with antibodies to GAPDH as a protein loading control