doi: 10.1038/nture07679 Emryonic Stem (ES) cell Hemngiolst Flk1 + Blst Colony 3 to 3.5 Dys 3-4 Dys ES differentition Sort of Flk1 + cells Supplementry Figure 1. Chrcteristion of lst colony development. Blst colonies were generted from Flk1 + cells isolted y FACS t dy 3-3.5 of ES cells differentition. The development of lst colonies ws studied during the 3-4 dys of culture. www.nture.com/nture 1
CD41 doi: 10.1038/nture07679 P3 Dy 4 P2 P4 P1 Hemtopoietic colonies per 30 000 cells 2000 1600 1200 800 400 0 Primitive Hemtopoiesis Definitive Hemtopoiesis P1 P2 P3 P4 Supplementry Figure 2. Hemtopoietic progenitors from Dy 4 Blst culture re restricted to the CD41 + popultions ) FACS nlysis of nd CD41 expression from Dy 4 Blst culture. P1: + CD41 -, P2: + CD41 +, P3: - CD41 + nd P4: - CD41 -. ) Numer of hemtopoietic (primitive nd definitive) colonies generted in methylcellulose y the FACS sorted P1, P2, P3 nd P4 popultions. Error rs indicte stndrd devition of the men (n=3). www.nture.com/nture 2
c-kit doi: 10.1038/nture07679 CD41- CD41+ P1 P5 P4 P3 P2 P6 Hemtopoietic colonies per 30 000 cells 3000 2500 2000 1500 1000 500 Primitive Hemtopoiesis Definitive Hemtopoiesis 0 P1 P2 P3 P4 P5 P6 Supplementry Figure 3. CD41+ popultions from Dy 2 Blst culture contin the mjority of hemtopoietic progenitors ) nd c-kit expression on CD41 - (left) nd CD41 + (right) cells of dy 2 Blst culture. P1: + c-kit + CD41 -, P2: + c-kit - CD41 -, P3: -/lo c-kit - CD41 -, P4: + c-kit + CD41 +, P5: -/lo c-kit + CD41 + nd P6: -/lo c-kit - CD41 + ) Numer of hemtopoietic (primitive nd definitive) colonies generted in methylcellulose y the FACS sorted P1, P2, P3, P4, P5 nd P6 popultions. Error rs indicte stndrd devition of the men (n=3). www.nture.com/nture 3
c-kit doi: 10.1038/nture07679 hi ckit + C+ Wter CD31 CD34 Endoglin Flk1 Flt1 VE-cdherin -Actin c DAPI CD31 DAPI CD41 DAPI CD31 CD41 Supplementry Figure 4. Loclistion of the endothelil hi c-kit + CD41 - popultion in lst colony. ) Semi-quntittive RT-PCR nlysis of expression of endothelil genes in sorted dy 2 hi c-kit + CD41 - cells. Positive control C+ corresponds to dy 4.5 EB. ) FACS nlysis of nd c-kit expression from CD41 - Dy 3 Blst culture. The red dots correspond to CD31 + cells. These cells re mostly + c-kit +. c) Immunostining of dy 3 lst colony. Left pnel is n overly of CD31 nd DAPI stinings. Middle pnel is n overly of CD41 nd DAPI stinings of the sme colony. Left pnel is n overly of CD31, CD41 nd DAPI stinings of the sme colony. Red: CD31, Green: CD41 nd Blue: DAPI. www.nture.com/nture 4
doi: 10.1038/nture07679 hi ckit + C+ Wter c Primitive Hemtopoiesis d Dy 1 Runx1 cmy Fli1 GATA2 Hex LMO2 Scl -Actin e T 0 + 1 dy T 0 + 2 dys Hemtopoietic colonies per 30 000 cells f 250 200 150 100 50 0 Definitive Hemtopoiesis CD41 + CD41 - Counts CD41 41.5% Frction of negtive wells 1.0 0.37 0.14 0.05 Frequency: 1/79 * 40 80 120 160 200 Numer of cells per well CFSE Supplementry Figure 5. Anlysis of hi c-kit + CD41 - popultion. ) Semi-quntittive RT-PCR nlysis of expression of erly hemtopoietic trnscription fctors in sorted hi c-kit + CD41 - cells. C+ corresponds to dy 4.5 EB. ) Pictures of sorted hi c-kit + CD41 - cells were tken fter 1 dy nd 2 dys of hemogenic endothelium culture. c) Numer of hemtopoietic colonies generted y CD41 + nd CD41 - cells sorted from dy 2 hemogenic endothelium culture. Error rs indicte stndrd devition of the men (n=3). d) FACS nlysis of CD41 expression nd level of CFSE fluorescence fter 1 dy of hemogenic endothelium culture. Top pnel is dot plot showing expression of CD41 together with CFSE fluorescence. Bottom pnel shows levels of CFSE fluorescence in CD41 + (old line, men fluorescence =321) nd CD41 - (norml line, men fluorescence =500) cells. e) Limiting dilution nlysis of the frequency of dy 2 hi c-kit + CD41 - cells giving rise to hemtopoietic cells. The frequency ws evluted to e 1 out of 79 cells in one experiment nd 1 out of 86 cells in nother experiment. f) My-Grunwld Giems stining of hemtopoietic cells generted fter 8 dys of culture y single hi c-kit + CD41 - cells. The presence of erythroid (*), mcrophge (rrow) nd neutrophil (open circle) cells is indicted. www.nture.com/nture 5
doi: 10.1038/nture07679 Counts Dy 1 Dy 2 Totl Totl 33.21% 0.6% hi ckit + CD41-0.1% Colonies per 30000 cells 3000 2500 2000 1500 1000 500 BL-CFC Assy Flk1 + hi ckit + CD41 - Brchyury-GFP Supplementry Figure 6. The hi c-kit + CD41 - popultion is different from the hemngiolst. ) FACS nlysis of expression of the mesoderm mrker Brchyury t dy 1, dy 2 of lst development (totl) nd y dy 2 hi c-kit + CD41 - cells (ottom pnel). ) Blst colony developmentl potentil of dy 3.5 EB Flk1 + nd hi c-kit + CD41 - popultions. Error rs indicte stndrd devition of the men (n=3). www.nture.com/nture 6
c-kit doi: 10.1038/nture07679 c-kit 10X c-kit 20X 20X c-kit 20X d d c-kit 40X 40X c-kit 40X c c-kit CD41 10X d d c-kit 40X 40X c-kit 40X d d E10.5 Cudl Prt e OP9 co-culture (+4 dys) 3.8 Supplementry Figure 7. + c-kit + CD41 - hemogenic endothelium cn e found in the cudl prt of E10.5 emryo. ) Immunostining of the dorsl ort (d) region for expression of c-kit nd t E10.5 (lens x10). Arrowhed indictes c-kit + cells. Blue: DAPI, Green: c-kit nd Red:. ) Immunostining of the dorsl ort for expression of c-kit nd t E10.5 (lens x20, first line nd lens x40, second nd third lines). Blue: DAPI, Green: c-kit nd Red:. c) Immunostining of the dorsl ort (d) region for expression of c-kit nd CD41 t E10.5 (lens x10). Arrowheds indicte c-kit + cells. Blue: DAPI, Green: c-kit nd Red: CD41. d) FACS nlysis of nd c-kit expression mong the CD41 - CD45 - cells isolted from the cudl prt of E10.5 emryo. e) After sorting of the + c-kit + CD41 - CD45 - cells, they were co-cultured on OP9 stroml cells for 4 dys. Round cells could e detected (rrow) nd generted hemtopoietic colonies upon replting (dt not shown). www.nture.com/nture 7
doi: 10.1038/nture07679 DAPI CD31 CD41 DAPI CD31 CD41 Supplementry Figure 8. Runx1 -/- tight dherent structures re composed of endothelil cells. Immunostining of dy 3 Runx1 -/- lst culture in dherent conditions. Left nd right pnels show overlys of CD31, CD41 nd DAPI stinings for two different colonies. Red: CD31, Green: CD41 nd Blue: DAPI. www.nture.com/nture 8
No Dox 0.1 μg/ml 1 μg/ml Wter doi: 10.1038/nture07679 No Dox 0.1 μg/ml 1 μg/ml c-kit 27.1 2.1 20.8 19.5 7.5 43.8 Runx1 69.5 1.3 47 12.7 31.4 17.3 CD11 CD41 cfms c-kit 7.8 21.4 18.8 21.5 41.8 9.5 Lysozyme -Actin 51.2 19.6 47 12.7 41 7.7 Supplementry Figure 9. Rescue y Runx1 of definitive hemtopoietic development. ) FACS nlysis of CD41/c-Kit (top) nd /c-kit expression (ottom) of Runx1 -/- irunx1 Dy 3 lst generted in sence, with 0.1 μg/ml nd 1 μg/ml of doxycycline (dox) dded 1 dy efore. ) Semi-quntittive RT-PCR nlysis of expression of Runx1 nd definitive hemtopoietic (myeloid) genes in the smples otined from ). www.nture.com/nture 9
doi: 10.1038/nture07679 Blst Colony Tight Colony 1200 Colonies per 10 5 cells 1000 800 600 400 200 0 Wild Type Runx1 -/- Supplementry Figure 10. Increse of tight colony numers in Runx1 -/- compred to wild type in BL-CFC ssy. ) Bright field pictures of representtive lst nd tight colonies. ) Numer of lst nd tight colonies generted from wild type nd Runx1 -/- Flk1 + cells. www.nture.com/nture 10
CD41 c-kit doi: 10.1038/nture07679 Wild type Hemngiolst Flk-1+ Tight Colony Blst Colony Runx1 -/- c d Immunofluorescence for nd c-kit DAPI FACS Anlysis For, c-kit nd CD41 6 54.5 Rescue Experiment with irunx1 Runx1-/- colonies 15.7 23.8 DAPI c-kit 3.5 12.8 Rescue 17.5 66 Supplementry Figure 11. Tight colonies generted from BL-CFC in sence of Runx1 contin hemogenic endothelium. ) Description of the model of Blst development. The sence of Runx1 leds to lock in the emergence of definitive hemtopoietic progenitors. ) Immunostining of dy 3 Runx1 -/- tight colony. Top pnel shows overlys of DAPI nd nd ottom pnel, DAPI nd c-kit. c) FACS nlysis of picked Dy 3 Runx1 -/- tight colonies for, c-kit nd CD41. d) Individul dy 3 inducile Runx1 Runx1 -/- tight colonies were trnsferred in one well with doxycycline. Pnel shows picture of well with hemtopoietic cells fter rescue of Runx1 -/- deficiency. www.nture.com/nture 11