Supplementary Figure 2 is related to Figure 3.

ScRNA-seq analysis of the BM from two representative mice engrafted with D17 EB cells 20 weeks after transplant and separation strategy for human and mouse cells in scRNA-Seq.

(A) UMAP embedding of a merged dataset of human cells in the two engrafted BM datasets (primary mice L1 and L5) colored by sample.

(B, C) Bone marrow chimerism of primary mouse L1 transplanted with D17 EB cells at 20 weeks. (B) Erythroid cells (hCD235a/hCD71) from total BM cells. (C) B cells (hIgM+/hCD10+), neutrophils (hCD33+/hCD14+), and T cells (hCD4+/hCD3+) from CD45+ cells.

(D) Stacked histogram of the relative proportions of the major hematopoietic cell populations detected by scRNAseq in the transplanted BM of L1 and L5 mice. Myeloid (M), Pro-erythroid cells (Pro-E), B cells, and T cells.

(E, G) Scatter plot of the number of genes detected in each cell on the grch38 and the mm10 assembly in the control dataset (E) and the engrafted mouse dataset (G)

(F, H) Violin Plot of the ratio of genes detected with the grch38 assembly and the mm10 assembly in the control dataset (F) and the engrafted mouse dataset (H)

(I, J) UMAP embedding of the cells profiled by scRNA-Seq in the control dataset (I) and the engrafted mouse dataset (J)

(K) UMAP embeddings of the cells in the engrafted mouse dataset before filtering out doublets.
A

Globin q-PCR analysis at single BFU-E colony level

<figure><img src="image_50.png" /><figcaption>Human BFU-E from CD34+ CB cells</figcaption></figure>

<figure><img src="image_51.png" /></figure>

B

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

qRT-PCR hCD19

<figure><img src="image_54.png" /></figure>

qRT-PCR hTCF7

<figure><img src="image_55.png" /></figure>

C

<figure><img src="image_56.png" /></figure>

D

<figure><img src="image_57.png" /></figure>

ΔCt
A

Globin q-PCR analysis at single BFU-E colony level

<figure><img src="image_54.png" /><figcaption>Human BFU-E from CD34+ CB cells</figcaption></figure>

<figure><img src="image_55.png" /></figure>

B

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

qRT-PCR hCD19

<figure><img src="image_58.png" /></figure>

qRT-PCR hTCF7

<figure><img src="image_59.png" /></figure>

C

<figure><img src="image_60.png" /></figure>

D

<figure><img src="image_61.png" /></figure>

ΔCt
Supplementary Figure 3 is related to Figure 4.

Molecular characterization of the grafted cells, globin RT-qPCR analysis from BFU-E at the single colony level.

(A) Up: Individual BFU-E colonies from BM cell cultures of primary recipients (approximately 18% of total colonies). Down: CD34+ human CB cells. Shown is the ratio of beta (blue), gamma (red) and epsilon (green) globin. CD34 CB cells give rise to colonies with a homogeneous ratio of beta- and gamma-globin, whereas BM cells give rise to a more diverse globin composition with a weak expression of epsilon-globin.

(B) qRT-PCR analysis for hCD19 (B lymphocyte surface antigen B4, B cell-specific marker) and hTCF7, a T-cell-specific marker, was performed on thymus, spleen, and bone marrow.

For each gene, delta Ct is the mean ± SEM of 3 independent experiments.

(C) Cytospins. May Grünwald-Giemsa staining of cells isolated from clonogenic assays in primary and secondary recipients. Scale bars represent 10µm.

(D) Representative qRT-PCR results obtained with a TaqMan® Array Human Hematopoiesis 96-well plate from hCD45+ BM cells.
Supplementary Figure 3 is related to Figure 4.

Molecular characterization of the grafted cells, globin RT-qPCR analysis from BFU-E at the single colony level.

(A) Up: Individual BFU-E colonies from BM cell cultures of primary recipients (approximately 18% of total colonies). Down: CD34+ human CB cells. Shown is the ratio of beta (blue), gamma (red) and epsilon (green) globin. CD34 CB cells give rise to colonies with a homogeneous ratio of beta- and gamma-globin, whereas BM cells give rise to a more diverse globin composition with a weak expression of epsilon-globin.

(B) qRT-PCR analysis for hCD19 (B lymphocyte surface antigen B4, B cell-specific marker) and hTCF7, a T-cell-specific marker, was performed on thymus, spleen, and bone marrow.

For each gene, delta Ct is the mean ± SEM of 3 independent experiments.

(C) Cytospins. May Grünwald-Giemsa staining of cells isolated from clonogenic assays in primary and secondary recipients. Scale bars represent 10µm.

(D) Representative qRT-PCR results obtained with a TaqMan® Array Human Hematopoiesis 96-well plate from hCD45+ BM cells.
Multilineage in saline-transplanted control

A

<figure><img src="image_58.png" /></figure>

Multilineage in CD34+ CB cells, secondary transplant

B

<figure><img src="image_59.png" /></figure>

NOD SCID primary recipients

<figure><img src="image_60.png" /></figure>

NOD SCID secondary recipients

<figure><img src="image_61.png" /></figure>

NOD SCID tertiary recipients

<figure><img src="image_62.png" /></figure>

D

C

E
Multilineage in saline-transplanted control

A

<figure><img src="image_62.png" /></figure>

Multilineage in CD34+ CB cells, secondary transplant

B

<figure><img src="image_63.png" /></figure>

NOD SCID primary recipients

<figure><img src="image_64.png" /></figure>

NOD SCID secondary recipients

<figure><img src="image_65.png" /></figure>

NOD SCID tertiary recipients

<figure><img src="image_66.png" /></figure>

D

C

E
Supplementary Figure 4 is related to Figure 5.

**Secondary transplants in NSG mice; representative recipients for saline and CD34⁺ CB cells and hematopoietic engraftment in primary, secondary and tertiary NOD-SCID recipients at 20 weeks post-graft.**

(A) Flow cytometric analysis of a representative control (saline-injected) secondary recipient. Myeloid (hCD33), B lymphoid (hCD19), T lymphoid (hCD3) and pro-erythroid (hCD71/hCD235a) lineages. Myeloid and lymphoid cells are analyzed from hCD45⁺ gated BM cells whereas Pro-E hCD71/hCD235a was analyzed on whole BM cells. (n=16, 8 independent experiments)

(B) Representative primary recipients transplanted with 7.10⁶ CD34⁺ CB cells (n= 13, 3 independent experiments). Same analysis as in (D).

(C) Primary NOD-SCID recipient inoculated with 4.10⁵ D17 EB cells (n=20, 3 independent experiments).

(D) Secondary NOD-SCID recipients inoculated with 7.10⁶ BM cells from primary recipients (n=16, 2 independent experiments).

(E) Tertiary NOD-SCID recipient inoculated with 7.10⁶ BM cells from secondary recipients (n=3, 1 experiment).

Data are presented as box plots and are mean ± SD or SEM according to the number of independent experiments. Wilcoxon Mann-Whitney test.
Supplementary Figure 4 is related to Figure 5.

Secondary transplants in NSG mice; representative recipients for saline and CD34+ CB cells
and hematopoietic engraftment in primary, secondary and tertiary NOD-SCID recipients at
20 weeks post-graft.

(A) Flow cytometric analysis of a representative control (saline-injected) secondary recipient. Myeloid (hCD33), B lymphoid (hCD19), T lymphoid (hCD3) and pro-erythroid (hCD71/hCD235a) lineages. Myeloid and lymphoid cells are analyzed from hCD45+ gated BM cells whereas Pro-E hCD71/hCD235a was analyzed on whole BM cells. (n=16, 8 independent experiments)

(B) Representative primary recipients transplanted with 7.10⁶ CD34+ CB cells (n= 13, 3 independent experiments). Same analysis as in (D).

(C) Primary NOD-SCID recipient inoculated with 4.10⁵ D17 EB cells (n=20, 3 independent experiments).

(D) Secondary NOD-SCID recipients inoculated with 7.10⁶ BM cells from primary recipients (n=16, 2 independent experiments).

(E) Tertiary NOD-SCID recipient inoculated with 7.10⁶ BM cells from secondary recipients (n=3, 1 experiment).

Data are presented as box plots and are mean ± SD or SEM according to the number of independent experiments. Wilcoxon Mann-Whitney test.
<figure><img src="image_63.png" /></figure>

D13

D15

D17

D19
<figure><img src="image_67.png" /></figure>

D13

D15

D17

D19
Supplementary Figure 5 is related to Figure 6.

Identification and evolution of the cell populations detected in the embryoid bodies by
scRNA-Seq at D13, 15, 17 and 19.

Repartition of the cells from each timepoint on the UMAP embedding of the EBs. EVT: Extravillous Trophoblast. MEP: Megakaryocyte-Erythroid Progenitors. ECM: Extracellular Matrix. AGM: Aorta-Gonad-Mesonephros.
Supplementary Figure 5 is related to Figure 6.

Identification and evolution of the cell populations detected in the embryoid bodies by
scRNA-Seq at D13, 15, 17 and 19.

Repartition of the cells from each timepoint on the UMAP embedding of the EBs. EVT: Extravillous Trophoblast. MEP: Megakaryocyte-Erythroid Progenitors. ECM: Extracellular Matrix. AGM: Aorta-Gonad-Mesonephros.
A

<figure><img src="image_64.png" /></figure>

B

<figure><img src="image_65.png" /></figure>

C

<figure><img src="image_66.png" /></figure>

<table><thead><tr><th></th><th>D0</th><th>D7</th><th>D13</th><th>D17</th><th>D19</th></tr></thead><tbody><tr><td>CFU/10 000 seeded cells</td><td>0</td><td>0.6 +/- 0.7</td><td>13 +/- 2.1</td><td>30.2 +/- 5.7</td><td>24.3 +/- 3.6</td></tr><tr><td>CFU/10 000 seeded CD34+ cells</td><td>0</td><td>1.8 +/- 2.1</td><td>82.7 +/- 13.4</td><td>203.8 +/- 38.4</td><td>198.2 +/- 29.4</td></tr></tbody></table>

D

<figure><img src="image_67.png" /></figure>

<table><thead><tr><th>Group</th><th>Lower</th><th>Estimate</th><th>Upper</th></tr></thead><tbody><tr><td>D13</td><td>195415</td><td>106723</td><td>58285</td></tr><tr><td>D15</td><td>39318</td><td>22258</td><td>12600</td></tr><tr><td>D17</td><td>1448</td><td>831</td><td>476</td></tr></tbody></table>
A

<figure><img src="image_68.png" /></figure>

B

<figure><img src="image_69.png" /></figure>

C

<figure><img src="image_70.png" /></figure>

<table><thead><tr><th></th><th>D0</th><th>D7</th><th>D13</th><th>D17</th><th>D19</th></tr></thead><tbody><tr><td>CFU/10 000 seeded cells</td><td>0</td><td>0.6 +/- 0.7</td><td>13 +/- 2.1</td><td>30.2 +/- 5.7</td><td>24.3 +/- 3.6</td></tr><tr><td>CFU/10 000 seeded CD34+ cells</td><td>0</td><td>1.8 +/- 2.1</td><td>82.7 +/- 13.4</td><td>203.8 +/- 38.4</td><td>198.2 +/- 29.4</td></tr></tbody></table>

D

<figure><img src="image_71.png" /></figure>

<table><thead><tr><th>Group</th><th>Lower</th><th>Estimate</th><th>Upper</th></tr></thead><tbody><tr><td>D13</td><td>19541.5</td><td>106723</td><td>58285</td></tr><tr><td>D15</td><td>39318</td><td>22258</td><td>12600</td></tr><tr><td>D17</td><td>1448</td><td>831</td><td>476</td></tr></tbody></table>
Supplementary Figure 6 is related to Figure 7.

Endothelial and hematopoietic potential of D0 to D19 EBs ex vivo.

EBs were assessed for their ability to form endothelial or hematopoietic colonies using
approved functional assays.

(A) Tubule formation in Matrigel (n=3) and flow cytometry with Ac LDL uptake and hCD31 as a marker (n=3). The highest EC differentiation was found between D7 and D13. **Scale bars represent 100µm.**

(B) Colony forming units from D0 to D19 EB cells (n=10). CFUs peaked at D17, but the number of hematopoietic cells remained low, indicating weak hematopoietic differentiation. **Scale bars represent 200µm**

(C) Histograms showing the frequency of CFUs from D0 to D19.

(D) LTC-IC assays were performed with D13 (n=1), D15 (n=1), and D17 (n=1) cells. Confidence intervals of 1/(LTC-IC cell frequency) were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution.
Supplementary Figure 6 is related to Figure 7.

Endothelial and hematopoietic potential of D0 to D19 EBs ex vivo.

EBs were assessed for their ability to form endothelial or hematopoietic colonies using
approved functional assays.

(A) Tubule formation in Matrigel (n=3) and flow cytometry with Ac LDL uptake and hCD31 as a marker (n=3). The highest EC differentiation was found between D7 and D13. Scale bars represent 100µm.

(B) Colony forming units from D0 to D19 EB cells (n=10). CFUs peaked at D17, but the number of hematopoietic cells remained low, indicating weak hematopoietic differentiation. Scale bars represent 200µm

(C) Histograms showing the frequency of CFUs from D0 to D19.

(D) LTC-IC assays were performed with D13 (n=1), D15 (n=1), and D17 (n=1) cells. Confidence intervals of 1/(LTC-IC cell frequency) were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution.
Supplemental Tables

<table><thead><tr><th rowspan="2">Combination N°</th><th colspan="10">Factor (ng/mL)</th><th colspan="10">Results</th></tr><tr><th>SCF<br>(37.38.39.<br>40.e.h)</th><th>TPO<br>(d)</th><th>FLT3 (37.38<br>.39.40)</th><th>BMP4<br>(39.40)</th><th>VEGF<br>(39)</th><th>IL3<br>(b.d.37.<br>40)</th><th>IL6<br>(b.d.f.38.<br>40)</th><th>IL1<br>β<br>(41)</th><th>GCSF<br>(b.37.<br>38.40)</th><th>IGF1<br>(36)</th><th>CD90<br>(e.f.<br>39.41)</th><th>CD109<br>(a)</th><th>CD13<br>(c.h)</th><th>CD117<br>(f.h.39)</th><th>CD38 (b.<br>d.h.36.37.39.<br>41)</th><th>HLA-<br>DR<br>(h.36.39<br>)</th><th>CD45</th><th>CD34 (a.b.<br>c.d.e.f.g.h.36.37.<br>38.39.40.41)</th><th>CD33<br>(c.h)</th><th>LTC-ICs at D16</th><th>Amplification<br>(Cells at<br>D17/seeded<br>iPSCs)</th></tr></thead><tbody><tr><td>1</td><td>300</td><td>10<br>0</td><td>20</td><td>20</td><td>200</td><td>5</td><td>50</td><td>50</td><td>10</td><td>50</td><td>2.39</td><td>0.1</td><td>0.07</td><td>0.44</td><td>0.1</td><td>0.06</td><td>0.41</td><td>0.94</td><td>0.1</td><td>11/1040000</td><td>3.01875</td></tr><tr><td>2</td><td>300</td><td>20</td><td>300</td><td>200</td><td>200</td><td>50</td><td>50</td><td>50</td><td>10</td><td>5</td><td>1.11</td><td>0.55</td><td>0.55</td><td>2.34</td><td>0.65</td><td>0.38</td><td>1.92</td><td>2.02</td><td>0.15</td><td>4/640000</td><td>1.7325</td></tr><tr><td>3</td><td>20</td><td>20</td><td>300</td><td>20</td><td>200</td><td>5</td><td>5</td><td>5</td><td>10</td><td>5</td><td>1.22</td><td>0.24</td><td>0.42</td><td>1.12</td><td>0.44</td><td>0.18</td><td>0.69</td><td>0.96</td><td>0.13</td><td>6/1390000</td><td>5.355</td></tr><tr><td>4</td><td>300</td><td>20</td><td>20</td><td>200</td><td>20</td><td>5</td><td>50</td><td>5</td><td>100</td><td>5</td><td>0.82</td><td>1.87</td><td>3.09</td><td>3.38</td><td>1.48</td><td>0.66</td><td>3.71</td><td>4.61</td><td>0.97</td><td>4/1340000</td><td>3.81675</td></tr><tr><td>5</td><td>20</td><td>10<br>0</td><td>20</td><td>20</td><td>200</td><td>50</td><td>50</td><td>50</td><td>100</td><td>5</td><td>1.77</td><td>1.97</td><td>2.66</td><td>5.24</td><td>2.9</td><td>1.01</td><td>3.58</td><td>5.48</td><td>2.2</td><td>6/1440000</td><td>6.23175</td></tr><tr><td>6</td><td>20</td><td>20</td><td>20</td><td>200</td><td>200</td><td>5</td><td>5</td><td>50</td><td>100</td><td>5</td><td>16.8<br>9</td><td>1.51</td><td>0.69</td><td>1.4</td><td>0.59</td><td>0.83</td><td>6.29</td><td>10.08</td><td>0.59</td><td>1/100000</td><td>0.462</td></tr><tr><td>7</td><td>20</td><td>10<br>0</td><td>300</td><td>20</td><td>20</td><td>5</td><td>50</td><td>5</td><td>10</td><td>5</td><td>3.44</td><td>0.28</td><td>0.15</td><td>1.05</td><td>0.31</td><td>0.24</td><td>0.51</td><td>1.14</td><td>0.23</td><td>1/1040000</td><td>3.78</td></tr><tr><td>8</td><td>300</td><td>20</td><td>20</td><td>20</td><td>200</td><td>50</td><td>5</td><td>5</td><td>10</td><td>50</td><td>0.92</td><td>1.54</td><td>2.71</td><td>3.47</td><td>1.39</td><td>0.62</td><td>2.62</td><td>4.27</td><td>0.63</td><td>7/1340000</td><td>3.92175</td></tr><tr><td>9</td><td>300</td><td>10<br>0</td><td>20</td><td>20</td><td>20</td><td>50</td><td>5</td><td>5</td><td>100</td><td>5</td><td>1.47</td><td>0.23</td><td>0.22</td><td>0.77</td><td>0.22</td><td>0.14</td><td>0.53</td><td>1.56</td><td>0.16</td><td>0</td><td>3.07125</td></tr><tr><td>10</td><td>20</td><td>20</td><td>20</td><td>200</td><td>20</td><td>50</td><td>50</td><td>5</td><td>10</td><td>50</td><td>5.22</td><td>0.48</td><td>0.48</td><td>0.68</td><td>0.75</td><td>0.2</td><td>3.99</td><td>1.48</td><td>0.14</td><td>0</td><td>1.6485</td></tr><tr><td>11</td><td>300</td><td>10<br>0</td><td>300</td><td>200</td><td>20</td><td>50</td><td>5</td><td>50</td><td>10</td><td>5</td><td>0.91</td><td>0.75</td><td>0.28</td><td>1.5</td><td>0.16</td><td>0.23</td><td>1.96</td><td>0.51</td><td>0.03</td><td>0</td><td>0.504</td></tr><tr><td>12</td><td>300</td><td>10<br>0</td><td>300</td><td>200</td><td>200</td><td>5</td><td>5</td><td>5</td><td>100</td><td>50</td><td>1.28</td><td>1.27</td><td>0.39</td><td>6.34</td><td>0.83</td><td>0.89</td><td>4.98</td><td>3.95</td><td>0.14</td><td>0</td><td>0.1785</td></tr><tr><td>13</td><td>20</td><td>20</td><td>300</td><td>20</td><td>20</td><td>50</td><td>5</td><td>50</td><td>100</td><td>50</td><td>1.78</td><td>0.34</td><td>0.17</td><td>0.88</td><td>0.19</td><td>0.11</td><td>0.35</td><td>1.45</td><td>0.31</td><td>3/90000</td><td>2.73</td></tr></tbody></table>
Supplemental Tables

<table><thead><tr><th rowspan="2">Combination N°</th><th colspan="10">Factor (ng/mL)</th><th colspan="10">Results</th></tr><tr><th>SCF<br>(37.38.39.<br>40.e.h)</th><th>TPO<br>(d)</th><th>FLT3 (37.38<br>.39.40)</th><th>BMP4<br>(39.40)</th><th>VEGF<br>(39)</th><th>IL3<br>(b.d.37.<br>40)</th><th>IL6<br>(b.d.f.38.<br>40)</th><th>IL1<br>β<br>(41)</th><th>GCSF<br>(b.37.<br>38.40)</th><th>IGF1<br>(36)</th><th>CD90<br>(e.f.<br>39.41)</th><th>CD109<br>(a)</th><th>CD13<br>(c.h)</th><th>CD117<br>(f.h.39)</th><th>CD38 (b.<br>d.h.36.37.39.<br>41)</th><th>HLA-<br>DR<br>(h.36.39<br>)</th><th>CD45</th><th>CD34 (a.b.<br>c.d.e.f.g.h.36.37.<br>38.39.40.41)</th><th>CD33<br>(c.h)</th><th>LTC-ICs at D16</th><th>Amplification<br>(Cells at<br>D17/seeded<br>iPSCs)</th></tr></thead><tbody><tr><td>1</td><td>300</td><td>10<br>0</td><td>20</td><td>20</td><td>200</td><td>5</td><td>50</td><td>50</td><td>10</td><td>50</td><td>2.39</td><td>0.1</td><td>0.07</td><td>0.44</td><td>0.1</td><td>0.06</td><td>0.41</td><td>0.94</td><td>0.1</td><td>11/1040000</td><td>3.01875</td></tr><tr><td>2</td><td>300</td><td>20</td><td>300</td><td>200</td><td>200</td><td>50</td><td>50</td><td>50</td><td>10</td><td>5</td><td>1.11</td><td>0.55</td><td>0.55</td><td>2.34</td><td>0.65</td><td>0.38</td><td>1.92</td><td>2.02</td><td>0.15</td><td>4/640000</td><td>1.7325</td></tr><tr><td>3</td><td>20</td><td>20</td><td>300</td><td>20</td><td>200</td><td>5</td><td>5</td><td>5</td><td>10</td><td>5</td><td>1.22</td><td>0.24</td><td>0.42</td><td>1.12</td><td>0.44</td><td>0.18</td><td>0.69</td><td>0.96</td><td>0.13</td><td>6/1390000</td><td>5.355</td></tr><tr><td>4</td><td>300</td><td>20</td><td>20</td><td>200</td><td>20</td><td>5</td><td>50</td><td>5</td><td>100</td><td>5</td><td>0.82</td><td>1.87</td><td>3.09</td><td>3.38</td><td>1.48</td><td>0.66</td><td>3.71</td><td>4.61</td><td>0.97</td><td>4/1340000</td><td>3.81675</td></tr><tr><td>5</td><td>20</td><td>10<br>0</td><td>20</td><td>20</td><td>200</td><td>50</td><td>50</td><td>50</td><td>100</td><td>5</td><td>1.77</td><td>1.97</td><td>2.66</td><td>5.24</td><td>2.9</td><td>1.01</td><td>3.58</td><td>5.48</td><td>2.2</td><td>6/1440000</td><td>6.23175</td></tr><tr><td>6</td><td>20</td><td>20</td><td>20</td><td>200</td><td>200</td><td>5</td><td>5</td><td>50</td><td>100</td><td>5</td><td>16.8<br>9</td><td>1.51</td><td>0.69</td><td>1.4</td><td>0.59</td><td>0.83</td><td>6.29</td><td>10.08</td><td>0.59</td><td>1/100000</td><td>0.462</td></tr><tr><td>7</td><td>20</td><td>10<br>0</td><td>300</td><td>20</td><td>20</td><td>5</td><td>50</td><td>5</td><td>10</td><td>5</td><td>3.44</td><td>0.28</td><td>0.15</td><td>1.05</td><td>0.31</td><td>0.24</td><td>0.51</td><td>1.14</td><td>0.23</td><td>1/1040000</td><td>3.78</td></tr><tr><td>8</td><td>300</td><td>20</td><td>20</td><td>20</td><td>200</td><td>50</td><td>5</td><td>5</td><td>10</td><td>50</td><td>0.92</td><td>1.54</td><td>2.71</td><td>3.47</td><td>1.39</td><td>0.62</td><td>2.62</td><td>4.27</td><td>0.63</td><td>7/1340000</td><td>3.92175</td></tr><tr><td>9</td><td>300</td><td>10<br>0</td><td>20</td><td>20</td><td>20</td><td>50</td><td>5</td><td>5</td><td>100</td><td>5</td><td>1.47</td><td>0.23</td><td>0.22</td><td>0.77</td><td>0.22</td><td>0.14</td><td>0.53</td><td>1.56</td><td>0.16</td><td>0</td><td>3.07125</td></tr><tr><td>10</td><td>20</td><td>20</td><td>20</td><td>200</td><td>20</td><td>50</td><td>50</td><td>5</td><td>10</td><td>50</td><td>5.22</td><td>0.48</td><td>0.48</td><td>0.68</td><td>0.75</td><td>0.2</td><td>3.99</td><td>1.48</td><td>0.14</td><td>0</td><td>1.6485</td></tr><tr><td>11</td><td>300</td><td>10<br>0</td><td>300</td><td>200</td><td>20</td><td>50</td><td>5</td><td>50</td><td>10</td><td>5</td><td>0.91</td><td>0.75</td><td>0.28</td><td>1.5</td><td>0.16</td><td>0.23</td><td>1.96</td><td>0.51</td><td>0.03</td><td>0</td><td>0.504</td></tr><tr><td>12</td><td>300</td><td>10<br>0</td><td>300</td><td>200</td><td>200</td><td>5</td><td>5</td><td>5</td><td>100</td><td>50</td><td>1.28</td><td>1.27</td><td>0.39</td><td>6.34</td><td>0.83</td><td>0.89</td><td>4.98</td><td>3.95</td><td>0.14</td><td>0</td><td>0.1785</td></tr><tr><td>13</td><td>20</td><td>20</td><td>300</td><td>20</td><td>20</td><td>50</td><td>5</td><td>50</td><td>100</td><td>50</td><td>1.78</td><td>0.34</td><td>0.17</td><td>0.88</td><td>0.19</td><td>0.11</td><td>0.35</td><td>1.45</td><td>0.31</td><td>3/90000</td><td>2.73</td></tr></tbody></table>
<table><tr><td>14</td><td>20</td><td>100</td><td>300</td><td>200</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>50</td><td>4.43</td><td>2.01</td><td>2.33</td><td>3.76</td><td>0.99</td><td>0.71</td><td>3.71</td><td>12.88</td><td>0.47</td><td>4/70000</td><td>0.336</td></tr><tr><td>15</td><td>300</td><td>20</td><td>300</td><td>20</td><td>20</td><td>5</td><td>50</td><td>50</td><td>100</td><td>50</td><td>1.91</td><td>0.45</td><td>0.39</td><td>0.82</td><td>0.19</td><td>0.2</td><td>0.8</td><td>1.8</td><td>0.28</td><td>9/1040000</td><td>3.07125</td></tr><tr><td>16</td><td>20</td><td>100</td><td>20</td><td>200</td><td>20</td><td>5</td><td>5</td><td>50</td><td>10</td><td>50</td><td>3.71</td><td>0.58</td><td>0.8</td><td>1.34</td><td>0.3</td><td>0.21</td><td>1.02</td><td>2.43</td><td>0.07</td><td>2/640000</td><td>1.87</td></tr></table>

## Supplemental Table 1 related to Figure 1.

Cytokine combinations and cytokine concentrations tested for their ability to promote hematopoietic differentiation. Results after 17 days in terms of cell phenotype, cell amplification and ability to generate LTC-ICs.

<table><thead><tr><th rowspan="3"></th><th colspan="10">Input (Concentrations in ng/mL)</th><th colspan="8">Output</th></tr><tr><th rowspan="2">SCF</th><th rowspan="2">TPO</th><th rowspan="2">FLT3</th><th rowspan="2">BMP4</th><th rowspan="2">VEGF</th><th rowspan="2">IL3</th><th rowspan="2">IL6</th><th rowspan="2">IL1</th><th rowspan="2">GCSF</th><th rowspan="2">IGF1</th><th colspan="2">% of CD34+</th><th colspan="2">% of CD43+</th><th colspan="2">% of CD45+</th><th colspan="2">% of CD117+</th></tr><tr><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th></tr></thead><tbody><tr><td>Combination A</td><td>24</td><td>21</td><td>21</td><td>194</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>5</td><td>3.11</td><td>7.49</td><td>4.25</td><td>10.24</td><td>2.24</td><td>4.17</td><td>4.97</td><td>9.47</td></tr><tr><td>Combination B</td><td>25</td><td>27</td><td>22</td><td>198</td><td>196</td><td>5</td><td>50</td><td>6</td><td>100</td><td>5</td><td>5.14</td><td>4.15</td><td>4.16</td><td>3.83</td><td>1.6</td><td>3.02</td><td>6.15</td><td>6.42</td></tr><tr><td>Combination C</td><td>22</td><td>20</td><td>300</td><td>22</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>50</td><td>6.89</td><td>8.53</td><td>4.58</td><td>3.42</td><td>3.99</td><td>15.72</td><td>7.02</td><td>2.82</td></tr></tbody></table>

## Supplemental Table 2 related to Figure 1.

Matrix-based three best combinations of cytokines and growth factors and the percentages of CD34+, CD43+, CD45+ and CD117+ obtained with the corresponding combinations.
<table><tr><td>14</td><td>20</td><td>100</td><td>300</td><td>200</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>50</td><td>4.43</td><td>2.01</td><td>2.33</td><td>3.76</td><td>0.99</td><td>0.71</td><td>3.71</td><td>12.88</td><td>0.47</td><td>4/70000</td><td>0.336</td></tr><tr><td>15</td><td>300</td><td>20</td><td>300</td><td>20</td><td>20</td><td>5</td><td>50</td><td>50</td><td>100</td><td>50</td><td>1.91</td><td>0.45</td><td>0.39</td><td>0.82</td><td>0.19</td><td>0.2</td><td>0.8</td><td>1.8</td><td>0.28</td><td>9/1040000</td><td>3.07125</td></tr><tr><td>16</td><td>20</td><td>100</td><td>20</td><td>200</td><td>20</td><td>5</td><td>5</td><td>50</td><td>10</td><td>50</td><td>3.71</td><td>0.58</td><td>0.8</td><td>1.34</td><td>0.3</td><td>0.21</td><td>1.02</td><td>2.43</td><td>0.07</td><td>2/640000</td><td>1.87</td></tr></table>

## Supplemental Table 1 related to Figure 1.

Cytokine combinations and cytokine concentrations tested for their ability to promote hematopoietic differentiation. Results after 17 days in terms of cell phenotype, cell amplification and ability to generate LTC-ICs.

<table><thead><tr><th rowspan="3"></th><th colspan="10">Input (Concentrations in ng/mL)</th><th colspan="8">Output</th></tr><tr><th rowspan="2">SCF</th><th rowspan="2">TPO</th><th rowspan="2">FLT3</th><th rowspan="2">BMP4</th><th rowspan="2">VEGF</th><th rowspan="2">IL3</th><th rowspan="2">IL6</th><th rowspan="2">IL1</th><th rowspan="2">GCSF</th><th rowspan="2">IGF1</th><th colspan="2">% of CD34+</th><th colspan="2">% of CD43+</th><th colspan="2">% of CD45+</th><th colspan="2">% of CD117+</th></tr><tr><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th><th>FD136-25</th><th>IMR90-16</th></tr></thead><tbody><tr><td>Combination A</td><td>24</td><td>21</td><td>21</td><td>194</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>5</td><td>3.11</td><td>7.49</td><td>4.25</td><td>10.24</td><td>2.24</td><td>4.17</td><td>4.97</td><td>9.47</td></tr><tr><td>Combination B</td><td>25</td><td>27</td><td>22</td><td>198</td><td>196</td><td>5</td><td>50</td><td>6</td><td>100</td><td>5</td><td>5.14</td><td>4.15</td><td>4.16</td><td>3.83</td><td>1.6</td><td>3.02</td><td>6.15</td><td>6.42</td></tr><tr><td>Combination C</td><td>22</td><td>20</td><td>300</td><td>22</td><td>200</td><td>50</td><td>50</td><td>5</td><td>100</td><td>50</td><td>6.89</td><td>8.53</td><td>4.58</td><td>3.42</td><td>3.99</td><td>15.72</td><td>7.02</td><td>2.82</td></tr></tbody></table>

## Supplemental Table 2 related to Figure 1.

Matrix-based three best combinations of cytokines and growth factors and the percentages of CD34+, CD43+, CD45+ and CD117+ obtained with the corresponding combinations.
<table><thead><tr><th>N°</th><th>hiPS</th><th>Cytometry/ qRT-PCR</th><th>Immunofluorescence</th><th>scRNA seq</th><th>Functional ex vivo assays</th><th>First recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th><th>Bone marrow scRNA seq</th><th>Second recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th><th>Third recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th></tr></thead></table>

## DOE

<table><tr><td>1</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>2</td><td>IMR90</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>3</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>9 (NOD SCID)</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>4</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>6 (NOD SCID)</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr></table>

## Kinetic experiments

<table><tr><td>5</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>6</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>7</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>8</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>9</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>10</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>11</td><td>PC1429</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>12</td><td>PCI-CAU</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr></table>

## Experiments with NOD SCID

<table><tr><td>13</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>0</td><td>No</td></tr><tr><td>14</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>8</td><td>Yes</td><td>No</td><td>8</td><td>Yes</td><td>0</td><td>No</td></tr><tr><td>15</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>8</td><td>Yes</td><td>No</td><td>8</td><td>Yes</td><td>3</td><td>Yes</td></tr><tr><td>16</td><td>CT-</td><td>No</td><td>No</td><td>No</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>3</td><td>Yes</td></tr></table>

## Experiments with NSG

<table><tr><td rowspan="4">17</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT-</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="2">18</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></table>
<table><thead><tr><th>N°</th><th>hiPS</th><th>Cytometry/ qRT-PCR</th><th>Immunofluorescence</th><th>scRNA seq</th><th>Functional ex vivo assays</th><th>First recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th><th>Bone marrow scRNA seq</th><th>Second recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th><th>Third recipients</th><th>Flow cytometry qRT-PCR hematopoietic assays</th></tr></thead></table>

## DOE

<table><tr><td>1</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>2</td><td>IMR90</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>3</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>9 (NOD SCID)</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>4</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>6 (NOD SCID)</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr></table>

## Kinetic experiments

<table><tr><td>5</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>6</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>7</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>8</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>9</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>10</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>11</td><td>PC1429</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>12</td><td>PCI-CAU</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td></tr></table>

## Experiments with NOD SCID

<table><tr><td>13</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>0</td><td>No</td></tr><tr><td>14</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>8</td><td>Yes</td><td>No</td><td>8</td><td>Yes</td><td>0</td><td>No</td></tr><tr><td>15</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>8</td><td>Yes</td><td>No</td><td>8</td><td>Yes</td><td>3</td><td>Yes</td></tr><tr><td>16</td><td>CT-</td><td>No</td><td>No</td><td>No</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>3</td><td>Yes</td></tr></table>

## Experiments with NSG

<table><tr><td rowspan="4">17</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT-</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="2">18</td><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></table>
<table><tbody><tr><td rowspan="6">19</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="2">20 LD A</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>30</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="4">21</td><td>LAM01.00 5</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>LAM02.00 2</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>LAM01.00 5</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="3">22</td><td>LAM02.00 2</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>17</td><td>Yes</td><td>Yes</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="2">23</td><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>10</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="2">24 AB G</td><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>0</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></tbody></table>

Cord blood sorted CD34+ cells

<table><tbody><tr><td>25</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>26</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>5</td><td>Yes</td><td>No</td><td>5</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>27</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></tbody></table>

Supplemental Table 3 related to Figure 2:

Table summarizing all the experiments performed, the number of animals and the hiPSC cell lines used.

CT: control.
<table><tbody><tr><td rowspan="6">19</td><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>FD136</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="2">20 LD A</td><td>PC1429</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>30</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="4">21</td><td>LAM01.00 5</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>LAM02.00 2</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>LAM01.00 5</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td rowspan="3">22</td><td>LAM02.00 2</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>3</td><td>Yes</td><td>No</td><td>3</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>1</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>17</td><td>Yes</td><td>Yes</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="2">23</td><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>10</td><td>Yes</td><td>No</td><td>0</td><td>No</td><td>No</td><td>No</td></tr><tr><td rowspan="2">24 AB G</td><td>CT -</td><td>No</td><td>No</td><td>No</td><td>No</td><td>2</td><td>Yes</td><td>No</td><td>0</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>PCI-CAU</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></tbody></table>

Cord blood sorted CD34+ cells

<table><tbody><tr><td>25</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>26</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>5</td><td>Yes</td><td>No</td><td>5</td><td>Yes</td><td>No</td><td>No</td></tr><tr><td>27</td><td>CD34+</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>4</td><td>Yes</td><td>No</td><td>4</td><td>Yes</td><td>No</td><td>No</td></tr></tbody></table>

Supplemental Table 3 related to Figure 2:

Table summarizing all the experiments performed, the number of animals and the hiPSC cell lines used.

CT: control.
<table><thead><tr><td>Number of cells</td><td>Number of recipients</td><td>Number of mice with >1% human cell chimerism</td></tr></thead><tbody><tr><td>100 000</td><td>6</td><td>6</td></tr><tr><td>50 000</td><td>6</td><td>6</td></tr><tr><td>10 000</td><td>6</td><td>3</td></tr><tr><td>5 000</td><td>6</td><td>1</td></tr><tr><td>1 000</td><td>6</td><td>0</td></tr></tbody></table>

**Supplemental Table 4 related to Figure 4**

Limiting dilution assay of D17 cells during primary transplantation.

Graded doses of one hundred thousand to one thousand D17 cells were transplanted into irradiated NSG mice. and the percentage of human CD45+ cells in the BM was analyzed 20 weeks after transplantation (n=6 mice per group). Confidence intervals of 1/(stem cell frequency) were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution. The limiting cord blood dilution assay published by Guo et al<sup>(i)</sup> served as a reference.
<table><thead><tr><td>Number of cells</td><td>Number of recipients</td><td>Number of mice with &gt;1% human cell chimerism</td></tr></thead><tbody><tr><td>100 000</td><td>6</td><td>6</td></tr><tr><td>50 000</td><td>6</td><td>6</td></tr><tr><td>10 000</td><td>6</td><td>3</td></tr><tr><td>5 000</td><td>6</td><td>1</td></tr><tr><td>1 000</td><td>6</td><td>0</td></tr></tbody></table>

**Supplemental Table 4 related to Figure 4**

Limiting dilution assay of D17 cells during primary transplantation.

Graded doses of one hundred thousand to one thousand D17 cells were transplanted into irradiated NSG mice. and the percentage of human CD45+ cells in the BM was analyzed 20 weeks after transplantation (n=6 mice per group). Confidence intervals of 1/(stem cell frequency) were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution. The limiting cord blood dilution assay published by Guo et al<sup>(i)</sup> served as a reference.
<table><thead><tr><th rowspan="2">Gene</th><th colspan="2">Endothelial (EB)</th><th colspan="2">Hematopoietic cells (EB)</th><th>Early AGM (CS10)</th><th>AGM (CS14-15)</th></tr><tr><th>APLNR+</th><th>GJA5+</th><th>HE</th><th>HSC candidate</th><th>HSC</th><th>HSC</th></tr></thead><tbody><tr><td>RUNX1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HOXA9</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MLLT3</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PTPRC</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SPN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>THY1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VNN2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HLF</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SPINK2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GFI1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SELP</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>STAT5A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ITGA4</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SVOPL</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>EMCN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ACE</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PROCR</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HOXB9</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LIN28B</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CSF1R</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL3RA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HLA-DRA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SELL</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MSI2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HEMGN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PROM1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CDH5</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IGFBP2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MEIS2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NRP2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SOX17</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GJA5</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL33</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DKK1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AGTR2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALDH1A1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CD44</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KCNK17</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BCL11A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LIN28A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GAD1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>FGF23</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Legend**

No expression

Very low expression/a few positive cells

Low to medium expression

Medium to high expression
<table><thead><tr><th rowspan="2">Gene</th><th colspan="2">Endothelial (EB)</th><th colspan="2">Hematopoietic cells (EB)</th><th>Early AGM (CS10)</th><th>AGM (CS14-15)</th></tr><tr><th>APLNR+</th><th>GJA5+</th><th>HE</th><th>HSC candidate</th><th>HSC</th><th>HSC</th></tr></thead><tbody><tr><td>RUNX1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HOXA9</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MLLT3</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PTPRC</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SPN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>THY1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VNN2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HLF</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SPINK2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GFI1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SELP</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>STAT5A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ITGA4</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SVOPL</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>EMCN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ACE</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PROCR</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HOXB9</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LIN28B</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CSF1R</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL3RA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HLA-DRA</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SELL</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MSI2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HEMGN</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PROM1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CDH5</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IGFBP2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MEIS2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NRP2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SOX17</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GJA5</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL33</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DKK1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>AGTR2</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ALDH1A1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CD44</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>KCNK17</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BCL11A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LIN28A</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>GAD1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>FGF23</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Legend**

No expression

Very low expression/a few positive cells

Low to medium expression

Medium to high expression
**Supplemental Table 5 related to Figure 6.**

Expression of markers for HSC ontogenesis in the endothelial and hematopoietic cells contained in D17 EBs described in Zeng et al., 2021⁵⁴ and Calvanese et al., 2022⁵³.

**Additional bibliography:**

a. Murray, L.J., Bruno, E., Uchida, N., Hoffman, R., Nayar, R., Yeo, E.L., Schuh, A.C., and Sutherland, D.R. (1999). CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells. Exp Hematol 27, 1282-1294. 10.1016/s0301-472x(99)00071-5.

b. Terstappen, L.W., Huang, S., Safford, M., Lansdorp, P.M., and Loken, M.R. (1991). Sequential generations of hematopoietic colonies derived from single lineage-committed CD34+CD38- progenitor cells. Blood 77, 1218-1227

c. Gaipa, G., Coustan-Smith, E., Todisco, E., Maglia, O., Biondi, A., and Campana, D. (2002). Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells. Haematologica 87, 347-356.

d. Yin, A.H., Miraglia, S., Zanjani, E.D., Almeida-Porada, G., Ogawa, M., Leary, A.G., Olweus, J., Kearney, J., and Buck, D.W. (1997). AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90, 5002-5012.

e. Radtke, S., Adair, J.E., Giese, M.A., Chan, Y.Y., Norgaard, Z.K., Enstrom, M., Haworth, K.G., Schefter, L.E., and Kiem, H.P. (2017). A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Sci Transl Med 9. 10.1126/scitranslmed.aan1145.

f. Murray, L., Chen, B., Galy, A., Chen, S., Tushinski, R., Uchida, N., Negrin, R., Tricot, G., Jagannath, S., Vesole, D., and et al. (1995). Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin- subpopulation from mobilized peripheral blood. Blood 85, 368-378.

g. Gunji, Y., Nakamura, M., Osawa, H., Nagayoshi, K., Nakauchi, H., Miura, Y., Yanagisawa, M., and Suda, T. (1993). Human primitive hematopoietic progenitor cells are more enriched in KITlow cells than in KIThigh cells. Blood 82, 3283-3289.
**Supplemental Table 5 related to Figure 6.**

Expression of markers for HSC ontogenesis in the endothelial and hematopoietic cells contained in D17 EBs described in Zeng et al., 2021⁵⁴ and Calvanese et al., 2022⁵³.

**Additional bibliography:**

a. Murray, L.J., Bruno, E., Uchida, N., Hoffman, R., Nayar, R., Yeo, E.L., Schuh, A.C., and Sutherland, D.R. (1999). CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells. Exp Hematol 27, 1282-1294. 10.1016/s0301-472x(99)00071-5.

b. Terstappen, L.W., Huang, S., Safford, M., Lansdorp, P.M., and Loken, M.R. (1991). Sequential generations of hematopoietic colonies derived from single lineage-committed CD34+CD38- progenitor cells. Blood 77, 1218-1227

c. Gaipa, G., Coustan-Smith, E., Todisco, E., Maglia, O., Biondi, A., and Campana, D. (2002). Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells. Haematologica 87, 347-356.

d. Yin, A.H., Miraglia, S., Zanjani, E.D., Almeida-Porada, G., Ogawa, M., Leary, A.G., Olweus, J., Kearney, J., and Buck, D.W. (1997). AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90, 5002-5012.

e. Radtke, S., Adair, J.E., Giese, M.A., Chan, Y.Y., Norgaard, Z.K., Enstrom, M., Haworth, K.G., Schefter, L.E., and Kiem, H.P. (2017). A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Sci Transl Med 9. 10.1126/scitranslmed.aan1145.

f. Murray, L., Chen, B., Galy, A., Chen, S., Tushinski, R., Uchida, N., Negrin, R., Tricot, G., Jagannath, S., Vesole, D., and et al. (1995). Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin- subpopulation from mobilized peripheral blood. Blood 85, 368-378.

g. Gunji, Y., Nakamura, M., Osawa, H., Nagayoshi, K., Nakauchi, H., Miura, Y., Yanagisawa, M., and Suda, T. (1993). Human primitive hematopoietic progenitor cells are more enriched in KITlow cells than in KIThigh cells. Blood 82, 3283-3289.
h. Gu, R., Wei, H., Wang, Y., Lin, D., Liu, B., Zhou, C., Liu, K., Gong, B., Wei, S., Zhang, G., et al. (2017). The number of CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells indicates post-chemotherapy hematopoietic recovery in patients with acute myeloid leukemia. PLoS One 12, e0180624. 10.1371/journal.pone.0180624.

i. Guo, B., Huang, X., Lee, M.R., Lee, S.A., and Broxmeyer, H.E. (2018). Antagonism of PPAR-gamma signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis. Nat Med 24, 360-367. 10.1038/nm.4477.
h. Gu, R., Wei, H., Wang, Y., Lin, D., Liu, B., Zhou, C., Liu, K., Gong, B., Wei, S., Zhang, G., et al. (2017). The number of CD34+CD38+CD117+HLA-DR+CD13+CD33+ cells indicates post-chemotherapy hematopoietic recovery in patients with acute myeloid leukemia. PLoS One 12, e0180624. 10.1371/journal.pone.0180624.

i. Guo, B., Huang, X., Lee, M.R., Lee, S.A., and Broxmeyer, H.E. (2018). Antagonism of PPAR-gamma signaling expands human hematopoietic stem and progenitor cells by enhancing glycolysis. Nat Med 24, 360-367. 10.1038/nm.4477.
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