# Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells

Olivier Piau, Mathias Brunet-Manquat, Laurence Petit, Bruno L'Homme, Brigitte Birebent, Christine Linard, Laetitia Moeckes, Thomas Zuliani, Hélène Lapillonne, Marc Benderitter, et al.

► To cite this version:

Olivier Piau, Mathias Brunet-Manquat, Laurence Petit, Bruno L'Homme, Brigitte Birebent, et al. Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells. Cell Stem Cell, 2023, 30 (12), pp.1610-1623.e7. 10.1016/j.stem.2023.11.002. hal-04355216

HAL Id: hal-04355216

https://cnrs.hal.science/hal-04355216

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# Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells

Olivier Piau, Mathias Brunet-Manquat, Laurence Petit, Bruno L'Homme, Brigitte Birebent, Christine Linard, Laetitia Moeckes, Thomas Zuliani, Hélène Lapillonne, Marc Benderitter, et al.

► To cite this version:

Olivier Piau, Mathias Brunet-Manquat, Laurence Petit, Bruno L'Homme, Brigitte Birebent, et al. Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells. Cell Stem Cell, 2023, 30 (12), pp.1610-1623.e7. 10.1016/j.stem.2023.11.002. hal-04355216

HAL Id: hal-04355216

https://cnrs.hal.science/hal-04355216v1

Submitted on 9 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

Olivier Piau<fnref n="1" />,<fnref n="2" />,§, Mathias Brunet-Manquat<fnref n="1" />,<fnref n="3" />,§, Bruno L'Homme<fnref n="4" />, Laurence Petit<fnref n="2" />, Brigitte Birebent<fnref n="3" />, Christine Linard<fnref n="4" />, Laetitia Moeckes<fnref n="5" />, Thomas Zuliani<fnref n="5" />, Hélène Lapillonne<fnref n="1" />,<fnref n="6" />, Marc Benderitter<fnref n="4" />, Luc Douay<fnref n="6" />, Alain Chapel<fnref n="1" />,<fnref n="4" />,§, Laurence Guyonneau-Harmand<fnref n="1" />,<fnref n="3" />,§,*,# and Thierry Jaffredo<fnref n="3" />,§,*

<footnote n="1">¹ Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, CRSA, F-75012 Paris, France.</footnote>

<footnote n="2">²Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris Seine, Laboratoire de Biologie du Développement/UMR7622, 9 Quai St-Bernard, 75005 Paris, France.</footnote>

<footnote n="3">³ EFS Ile de France, Unité d'Ingénierie et de Thérapie Cellulaire, Créteil, F-94017, France.</footnote>

<footnote n="4">⁴ Laboratoire de radiobiologie des expositions médicales (LRMed), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), F-92262 Fontenay-aux-Roses, France.</footnote>

<footnote n="5">⁵ Etablissement Français du Sang – Atlantic Bio GMP – 2, rue Aronnax 44800 Saint-Herblain, France.</footnote>

<footnote n="6">⁶ AP-HP, Hôpital St Antoine/Trousseau, Service d'Hématologie Biologique, F-75012 Paris, France.</footnote>

<footnote>*Correspondence to: laurence.harmand@upmc.fr; thierry.jaffredo@upmc.fr</footnote>

<footnote>#Lead Contact Laurence Guyonneau-Harmand; laurence.harmand@upmc.fr</footnote>

<footnote n="5">⁵These authors contributed equally to this work</footnote>

<footnote>§Co-senior authors</footnote>
<figure><img src="image_2.png" /></figure>

Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

Olivier Piau<fnref n="1" />,<fnref n="2" />,§, Mathias Brunet-Manquat<fnref n="1" />,<fnref n="3" />,§, Bruno L'Homme<fnref n="4" />, Laurence Petit<fnref n="2" />, Brigitte Birebent<fnref n="3" />, Christine Linard<fnref n="4" />, Laetitia Moeckes<fnref n="5" />, Thomas Zuliani<fnref n="5" />, Hélène Lapillonne<fnref n="1" />,<fnref n="6" />, Marc Benderitter<fnref n="4" />, Luc Douay<fnref n="6" />, Alain Chapel<fnref n="1" />,<fnref n="4" />,§, Laurence Guyonneau-Harmand<fnref n="1" />,<fnref n="3" />,§,*,# and Thierry Jaffredo<fnref n="3" />,§,*

<footnote n="1">¹Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine, CRSA, F-75012 Paris, France.</footnote>

<footnote n="2">²Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris Seine, Laboratoire de Biologie du Développement/UMR7622, 9 Quai St-Bernard, 75005 Paris, France.</footnote>

<footnote n="3">³ EFS Ile de France, Unité d'Ingénierie et de Thérapie Cellulaire, Créteil, F-94017, France.</footnote>

<footnote n="4">⁴ Laboratoire de radiobiologie des expositions médicales (LRMed), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), F-92262 Fontenay-aux-Roses, France.</footnote>

<footnote n="5">⁵ Etablissement Français du Sang – Atlantic Bio GMP – 2, rue Aronnax 44800 Saint-Herblain, France.</footnote>

<footnote n="6">⁶ AP-HP, Hôpital St Antoine/Trousseau, Service d'Hématologie Biologique, F-75012 Paris, France.</footnote>

<footnote>*Correspondence to: laurence.harmand@upmc.fr; thierry.jaffredo@upmc.fr</footnote>

<footnote>#Lead Contact Laurence Guyonneau-Harmand; laurence.harmand@upmc.fr</footnote>

<footnote n="5">⁵These authors contributed equally to this work</footnote>

<footnote>§Co-senior authors</footnote>
## SUMMARY:

Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage, hematopoietic population that can be serially transplanted. Single cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSC and will help identify key regulators of HSC production during human ontogeny.

## INTRODUCTION

Despite intensive efforts over the last decades, vector-free, transplantable human HSCs have not yet been successfully generated. In contrast, many blood cell types useful for human therapies have been efficiently generated from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), including erythroid cells ¹⁻⁴, B ⁵,⁶ and T cells ⁷⁻¹⁰, myeloid cells ¹¹, megakaryocytes ¹²,¹³, or hematopoietic progenitor cells ¹⁴. In recent years, many studies have sought to derive a population of CD34⁺CD45⁺ cells from pluripotent stem cells, with a phenotype approaching that of adult HSCs ¹⁵⁻¹⁹ used in clinical transplantation. These include reprogramming of adult cells by forced expression of transcription factors in fibroblasts ²⁰ or endothelial cells ²¹. However, these CD34⁺CD45⁺ populations have mainly demonstrated poor transplantation potential in immunocompromised mice, undermining therapeutic use. The only reports of successful generation of bona fide HSCs were obtained by redirecting hiPSC-derived or primary human endothelial cells with transcription factors ²²⁻²⁴. However, the introduction of plasmids encoding oncogenes or the use of feeder cells precludes the use of these reprogrammed cells for clinical applications.
## SUMMARY:

Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage, hematopoietic population that can be serially transplanted. Single cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSC and will help identify key regulators of HSC production during human ontogeny.

## INTRODUCTION

Despite intensive efforts over the last decades, vector-free, transplantable human HSCs have not yet been successfully generated. In contrast, many blood cell types useful for human therapies have been efficiently generated from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), including erythroid cells ¹⁻⁴, B ⁵,⁶ and T cells ⁷⁻¹⁰, myeloid cells ¹¹, megakaryocytes ¹²,¹³, or hematopoietic progenitor cells ¹⁴. In recent years, many studies have sought to derive a population of CD34⁺CD45⁺ cells from pluripotent stem cells, with a phenotype approaching that of adult HSCs ¹⁵⁻¹⁹ used in clinical transplantation. These include reprogramming of adult cells by forced expression of transcription factors in fibroblasts ²⁰ or endothelial cells ²¹. However, these CD34⁺CD45⁺ populations have mainly demonstrated poor transplantation potential in immunocompromised mice, undermining therapeutic use. The only reports of successful generation of bona fide HSCs were obtained by redirecting hiPSC-derived or primary human endothelial cells with transcription factors ²²⁻²⁴. However, the introduction of plasmids encoding oncogenes or the use of feeder cells precludes the use of these reprogrammed cells for clinical applications.
Interestingly, transplantable human HSCs can be obtained in teratomas, albeit with low frequency <sup>25-27</sup>. While not directly applicable to human therapy, this demonstrates that generation of HSCs from human pluripotent stem cells is possible and that cell-cell interactions play a key role in promoting self-renewal and multilineage repopulation activity <sup>26</sup>. Teratomas recapitulate some aspects of embryonic development. Although this has not been specifically investigated, the presence of HSCs argues for mechanisms reminiscent of early hematopoietic development. Indeed, the first transplantable HSCs arise from a specialized population of endothelial cells, namely hemogenic endothelial cells, formed early during embryonic development in the aorta-gonad-mesonephros (AGM) region <sup>28,29</sup>, through an endothelial-hematopoietic transition (EHT) <sup>30,31</sup>. Some of these steps are likely recapitulated in embryoid body (EB) cultures that mimic events during hematopoiesis development. In particular, the formation of the hemogenic endothelium and budding of HCs are well documented <sup>32-34</sup>.

Here, we describe a vector-free and stroma-free system for generating human HSCs capable of robust, long-term multipotent reconstitution and self-renewal in vivo, from hiPSCs. This protocol uses EB formation to generate a cell population resembling nascent HSCs found in the human embryonic AGM. Overall, our results demonstrate that the generation of feeder-and vector-free human HSCs is now feasible and is achieved by producing cells that more closely resemble the original HSCs that emerge during ontogeny.

## RESULTS

### Establishment of culture conditions

The optimal culture conditions to generate HSCs were established using a Design of Experiment approach (DOE). DOE is a statistical method for improving process performance by determining the optimal combination of input factors to optimize the output parameters, using the concept of desirability <sup>35</sup>. We first performed a literature review on *in vitro* HSC differentiation, amplification and maintenance (Figure 1). Then, an ensemble of 10 cytokines and growth factors reported to be important at different stages of mesoderm and hematopoietic specification <sup>36-41</sup> was defined. These cytokines and growth factors are (i) bone morphogenetic protein 4 (BMP-4), vascular endothelial growth factor (VEGF), and insulin-like
Interestingly, transplantable human HSCs can be obtained in teratomas, albeit with low frequency <sup>25-27</sup>. While not directly applicable to human therapy, this demonstrates that generation of HSCs from human pluripotent stem cells is possible and that cell-cell interactions play a key role in promoting self-renewal and multilineage repopulation activity <sup>26</sup>. Teratomas recapitulate some aspects of embryonic development. Although this has not been specifically investigated, the presence of HSCs argues for mechanisms reminiscent of early hematopoietic development. Indeed, the first transplantable HSCs arise from a specialized population of endothelial cells, namely hemogenic endothelial cells, formed early during embryonic development in the aorta-gonad-mesonephros (AGM) region <sup>28,29</sup>, through an endothelial-hematopoietic transition (EHT) <sup>30,31</sup>. Some of these steps are likely recapitulated in embryoid body (EB) cultures that mimic events during hematopoiesis development. In particular, the formation of the hemogenic endothelium and budding of HCs are well documented <sup>32-34</sup>.

Here, we describe a vector-free and stroma-free system for generating human HSCs capable of robust, long-term multipotent reconstitution and self-renewal in vivo, from hiPSCs. This protocol uses EB formation to generate a cell population resembling nascent HSCs found in the human embryonic AGM. Overall, our results demonstrate that the generation of feeder-and vector-free human HSCs is now feasible and is achieved by producing cells that more closely resemble the original HSCs that emerge during ontogeny.

## RESULTS

### Establishment of culture conditions

The optimal culture conditions to generate HSCs were established using a Design of Experiment approach (DOE). DOE is a statistical method for improving process performance by determining the optimal combination of input factors to optimize the output parameters, using the concept of desirability <sup>35</sup>. We first performed a literature review on *in vitro* HSC differentiation, amplification and maintenance (Figure 1). Then, an ensemble of 10 cytokines and growth factors reported to be important at different stages of mesoderm and hematopoietic specification <sup>36-41</sup> was defined. These cytokines and growth factors are (i) bone morphogenetic protein 4 (BMP-4), vascular endothelial growth factor (VEGF), and insulin-like
growth factor-1 (IGF-1), (ii) FMS-like tyrosine kinase ligand (FLT3-L), stem cell factor (SCF), interleukin-1 (IL1), interleukin-3 (IL3), interleukin-6 (IL6), granulocyte colony-stimulating factor (G-CSF), and thrombopoietin (TPO). The time frame of the protocol was set to 17 days (D17), according to Lengerke and colleagues⁴⁰. We defined a concentration range for each of the cytokines as an input variable (Table S1). As output variables, we chose cell amplification, the frequency of long-term culture-initiating cells (LTC-ICs) and the expression of several surface markers associated with populations amenable to long-term transplantation, i.e., CD34, CD38, CD71, CD109, HLA-DR, CD90, CD33, CD13, CD117 (Table S1). The software defined sixteen different combinations, which were tested by measuring baseline variables (Table S1). These results were then subjected to regression analysis, and the response was plotted. The predictive model identified three efficient combinations of cytokines (Table S2). These three combinations were used to differentiate the FD136-25 and IMR90-16 hiPSC lines for 17 days. Each of the combinations triggered cell proliferation, low hematopoietic commitment (CD34⁺CD45⁺ and/or CD117⁺CD90⁺). Flow cytometric analysis of cell populations grown under the three conditions was performed using a panel of endothelial and hematopoietic-specific markers (Figure 1 and Table S2). Combination A showed the best ratio of immature (CD34⁺, CD43⁺, and CD117⁺) to mature (CD45⁺) hematopoietic markers. Differentiated FD136-25 cells were also grafted in irradiated NOD-SCID primary recipients (Figure 1 and Table S2). Combination A also showed the highest rate of human CD45⁺ cells in NOD/SCID-LtSz-scid/scid (NOD/SCID) mice and was therefore selected for further use (Figure 1).

**Long-term, multilineage repopulating activity and phenotypic analysis of the hematopoietic populations**

While CD34⁺CD45⁺ HCs appeared to burst from embryoid bodies (EBs) at D10 to D14 in standard protocols for hiPSC differentiation, ¹⁸,²²,⁴⁰,⁴², the EBs in combination A at D17 remained compact and spherical without bursting, indicating a delay in differentiation (Figure 2A). The D17 EB hematopoietic repopulation potential was tested by transplanting 4.10⁵ dissociated cells retro-orbitally into 8-week-old sublethally irradiated (2.4-3.5 Gy) NOD.Cg-PrkdcscidІІ2rgtm1Wjl/SzJ (NSG) mice for 20 weeks (Figure 2A). The efficiency of reconstitution was systematically compared to that obtained with cord blood (CB)-derived CD34⁺ cells. These culture conditions were reproducibly and successfully applied to four additional individual
growth factor-1 (IGF-1), (ii) FMS-like tyrosine kinase ligand (FLT3-L), stem cell factor (SCF), interleukin-1 (IL1), interleukin-3 (IL3), interleukin-6 (IL6), granulocyte colony-stimulating factor (G-CSF), and thrombopoietin (TPO). The time frame of the protocol was set to 17 days (D17), according to Lengerke and colleagues⁴⁰. We defined a concentration range for each of the cytokines as an input variable (Table S1). As output variables, we chose cell amplification, the frequency of long-term culture-initiating cells (LTC-ICs) and the expression of several surface markers associated with populations amenable to long-term transplantation, i.e., CD34, CD38, CD71, CD109, HLA-DR, CD90, CD33, CD13, CD117 (Table S1). The software defined sixteen different combinations, which were tested by measuring baseline variables (Table S1). These results were then subjected to regression analysis, and the response was plotted. The predictive model identified three efficient combinations of cytokines (Table S2). These three combinations were used to differentiate the FD136-25 and IMR90-16 hiPSC lines for 17 days. Each of the combinations triggered cell proliferation, low hematopoietic commitment (CD34⁺CD45⁺ and/or CD117⁺CD90⁺). Flow cytometric analysis of cell populations grown under the three conditions was performed using a panel of endothelial and hematopoietic-specific markers (Figure 1 and Table S2). Combination A showed the best ratio of immature (CD34⁺, CD43⁺, and CD117⁺) to mature (CD45⁺) hematopoietic markers. Differentiated FD136-25 cells were also grafted in irradiated NOD-SCID primary recipients (Figure 1 and Table S2). Combination A also showed the highest rate of human CD45⁺ cells in NOD/SCID-LtSz-scid/scid (NOD/SCID) mice and was therefore selected for further use (Figure 1).

**Long-term, multilineage repopulating activity and phenotypic analysis of the hematopoietic populations**

While CD34⁺CD45⁺ HCs appeared to burst from embryoid bodies (EBs) at D10 to D14 in standard protocols for hiPSC differentiation, ¹⁸,²²,⁴⁰,⁴², the EBs in combination A at D17 remained compact and spherical without bursting, indicating a delay in differentiation (Figure 2A). The D17 EB hematopoietic repopulation potential was tested by transplanting 4.10⁵ dissociated cells retro-orbitally into 8-week-old sublethally irradiated (2.4-3.5 Gy) NOD.Cg-PrkdcscidІІ2rgtm1Wjl/SzJ (NSG) mice for 20 weeks (Figure 2A). The efficiency of reconstitution was systematically compared to that obtained with cord blood (CB)-derived CD34⁺ cells. These culture conditions were reproducibly and successfully applied to four additional individual
hiPSC lines obtained with different reprogramming protocols (Figure 2A; Table S3, Materials and Methods), demonstrating the robustness of the method. The presence of human HCs in mouse bone marrow (BM) was monitored by flow cytometry, functional hematopoietic assays (T-cell activation and hemoglobin switch), cytological assays, RT-qPCR, and single-cell RNA sequencing (scRNA-Seq) (Table S3).

Primary NSG recipients transplanted with D17 cells derived from one or the other of the five hiPSC lines (Table S3) exhibited reconstitution of human hematopoietic cells (59/60), as shown by flow cytometric analysis of human (h) CD45 (Figure 2B) and surface expression of hCD34, hCD43, and hCD45 in mouse BMs (Figure 2C, D). The five hiPSC lines tested showed overall efficient hematopoietic reconstitution, with a mean of 4.1±0.6% CD34+, 7.2±1.0% CD43+, and 13.3±1.5% hCD45+ cells in mouse total mononucleated cells BM (Figure 2C, D). This is more than 130-fold the threshold of 0.1% hCD45+ normally considered positive for transplantation of human HCs into NSG mice<sup>43</sup>. These grafting results were similar to those obtained with CD34<sup>+</sup> CB cells under the same conditions (Figure 2D). To test the robustness of the protocol, Pci-CAU hiPSCs were differentiated by an independent laboratory (Atlantic Bio GMP Platforme, Nantes; France), inoculated into irradiated NSG mice, and monitored over time for up to 20 weeks (Figure S1A-C). These cells showed robust engraftment and multilineage differentiation, similar to cells produced in the original laboratory (compare Figure S1C, 20 weeks with Figure 2D). This confirms the reproducibility of the protocol.

Twenty weeks after transplantation, the mouse BM harbors robust human myeloid, B- and T-lymphoid cells (hCD33<sup>+</sup>, hCD19<sup>+</sup> and hCD3<sup>+</sup>, respectively) and hCD235a<sup>+</sup> erythroid progenitors/precursors (Figure 2E) <sup>44-46</sup>. Figure 2F shows a representative cytometry analysis of BMs isolated from NSG primary recipients transplanted with D17 EB cells, mock-injected (saline, Figure S1D) or CD34<sup>+</sup> CB cells (Figure S1E). The D17 EB cells (Figure 2F) show a similar multilineage pattern as the CD34<sup>+</sup> CB cells (Figure S1E), including myeloid (hCD14<sup>+</sup>hCD15<sup>+</sup>), B lymphoid (hCD19<sup>+</sup>hIgM<sup>+</sup>), T lymphoid (hCD3<sup>+</sup>) and red blood cells (hCD71<sup>+</sup>hCD235a<sup>+</sup>). We also analyzed the primitive hCD34<sup>+</sup>hCD38<sup>+</sup> or hCD38<sup>-</sup> populations in the hCD45<sup>+</sup> cell population in 14 primary mice (2 independent series) inoculated with D17 EB cells. The total percentage of hCD45<sup>+</sup> cells was 5.2±1.5% (Figure S1F). The percentages of hCD45<sup>+</sup>hCD34<sup>+</sup>hCD38<sup>+</sup> cells were 4.9±0.7% and that of hCD45<sup>+</sup>hCD34<sup>+</sup>hCD38<sup>-</sup> cells was 0.5±0.2% (Figure S1G, H). These numbers
hiPSC lines obtained with different reprogramming protocols (Figure 2A; Table S3, Materials and Methods), demonstrating the robustness of the method. The presence of human HCs in mouse bone marrow (BM) was monitored by flow cytometry, functional hematopoietic assays (T-cell activation and hemoglobin switch), cytological assays, RT-qPCR, and single-cell RNA sequencing (scRNA-Seq) (Table S3).

Primary NSG recipients transplanted with D17 cells derived from one or the other of the five hiPSC lines (Table S3) exhibited reconstitution of human hematopoietic cells (59/60), as shown by flow cytometric analysis of human (h) CD45 (Figure 2B) and surface expression of hCD34, hCD43, and hCD45 in mouse BMs (Figure 2C, D). The five hiPSC lines tested showed overall efficient hematopoietic reconstitution, with a mean of 4.1±0.6% CD34+, 7.2±1.0% CD43+, and 13.3±1.5% hCD45+ cells in mouse total mononucleated cells BM (Figure 2C, D). This is more than 130-fold the threshold of 0.1% hCD45+ normally considered positive for transplantation of human HCs into NSG mice<sup>43</sup>. These grafting results were similar to those obtained with CD34<sup>+</sup> CB cells under the same conditions (Figure 2D). To test the robustness of the protocol, Pci-CAU hiPSCs were differentiated by an independent laboratory (Atlantic Bio GMP Platforme, Nantes; France), inoculated into irradiated NSG mice, and monitored over time for up to 20 weeks (Figure S1A-C). These cells showed robust engraftment and multilineage differentiation, similar to cells produced in the original laboratory (compare Figure S1C, 20 weeks with Figure 2D). This confirms the reproducibility of the protocol.

Twenty weeks after transplantation, the mouse BM harbors robust human myeloid, B- and T-lymphoid cells (hCD33<sup>+</sup>, hCD19<sup>+</sup> and hCD3<sup>+</sup>, respectively) and hCD235a<sup>+</sup> erythroid progenitors/precursors (Figure 2E) <sup>44-46</sup>. Figure 2F shows a representative cytometry analysis of BMs isolated from NSG primary recipients transplanted with D17 EB cells, mock-injected (saline, Figure S1D) or CD34<sup>+</sup> CB cells (Figure S1E). The D17 EB cells (Figure 2F) show a similar multilineage pattern as the CD34<sup>+</sup> CB cells (Figure S1E), including myeloid (hCD14<sup>+</sup>hCD15<sup>+</sup>), B lymphoid (hCD19<sup>+</sup>hIgM<sup>+</sup>), T lymphoid (hCD3<sup>+</sup>) and red blood cells (hCD71<sup>+</sup>hCD235a<sup>+</sup>). We also analyzed the primitive hCD34<sup>+</sup>hCD38<sup>+</sup> or hCD38<sup>-</sup> populations in the hCD45<sup>+</sup> cell population in 14 primary mice (2 independent series) inoculated with D17 EB cells. The total percentage of hCD45<sup>+</sup> cells was 5.2±1.5% (Figure S1F). The percentages of hCD45<sup>+</sup>hCD34<sup>+</sup>hCD38<sup>+</sup> cells were 4.9±0.7% and that of hCD45<sup>+</sup>hCD34<sup>+</sup>hCD38<sup>-</sup> cells was 0.5±0.2% (Figure S1G, H). These numbers
are consistent with works reporting transplantation of CD34⁺ CB cells into NSG mice at 20 weeks of age ²³, ⁴⁷.

Peripheralization of the transplanted cells was confirmed by the presence of hCD45⁺ cells in the circulating blood, which showed a multilineage pattern (Figure S1G) resembling that observed in the peripheral blood of CD34⁺ CB transplanted recipients (Figure S1H), whereas no signal was found in mock-injected animals (Figure S1F). In the thymus, hCD3⁺ cells showed TCRαβ expression greater than 60% and TCRγδ expression less than 30% (Figure 2G and S1I), a pattern consistent with that obtained with CD34⁺ CB cells (Figure S1J) and confirming the presence of mature T cells as demonstrated by the expression of hCD4 and hCD8 (Figure 2G) ⁴⁸. In addition, hCD45⁺ cells in the spleen were characterized by the presence of T and B cells expressing hTCRβ and hCD19, respectively (Figure S1K), similar to CD34⁺ CB grafts (Figure S1L). Figure 2H shows multilineage analysis of representative samples from three different graft series, demonstrating lineage variability between individuals.

## Single-cell transcriptome and functional analysis of the grafted mouse BM

To get an unbiased view of the human cells in the mice BM after engraftment, we performed 10X Genomics on the BM of two representative primary mice (L5 and L1) 20 weeks after transplantation. As shown in Figure 3A-C and in Figure S2A, the BM of both recipients contained human erythroid, B, T, plasmacytoid dendritic, monocytic, and MAST cells (the strategy for distinguishing human and mouse cells is described in detail in the Materials and Methods section). The proportion of the different cell types was consistent between scRNA-Seq and FACS analysis and between the two mice (Figures 3D-F and Figure S2A-D). Human erythroid cells expressed α-, β-, γ-, and δ-globins, indicating mature erythropoiesis (Figure 3G).

To substantiate the functionality of the transplanted cells, we performed a human-selective clonogenic assay using BM cells isolated from primary recipients. We obtained an overall frequency of 17.5±2.4 colonies from 10⁴ BM cells (Figure 4A), (to be compared with 19.2±3.8 colonies from 10⁴ total BM cells obtained for the CD34 primary recipients). These colonies were divided into CFU-GEMM (Figure 4A and 4B1), BFU-E (Figure 4B2), and CFU-GM colonies (Figure 4B3). Cytological analysis of colonies revealed the presence of mature macrophages (Figure 4C1), histiomonocytes (Figure 4C2), and erythroblasts (Figure 4C3). We also analyzed the ability of human erythroid progenitor cells isolated from mouse BM to
are consistent with works reporting transplantation of CD34+ CB cells into NSG mice at 20 weeks of age<sup>23, 47</sup>.

Peripheralization of the transplanted cells was confirmed by the presence of hCD45+
cells in the circulating blood, which showed a multilineage pattern (Figure S1G) resembling
that observed in the peripheral blood of CD34+ CB transplanted recipients (Figure S1H),
whereas no signal was found in mock-injected animals (Figure S1F). In the thymus, hCD3+ cells
showed TCRαβ expression greater than 60% and TCRγδ expression less than 30% (Figure 2G
and S1I), a pattern consistent with that obtained with CD34+ CB cells (Figure S1J) and
confirming the presence of mature T cells as demonstrated by the expression of hCD4 and
hCD8 (Figure 2G) <sup>48</sup>. In addition, hCD45+ cells in the spleen were characterized by the presence
of T and B cells expressing hTCRβ and hCD19, respectively (Figure S1K), similar to CD34+ CB
grafts (Figure S1L). Figure 2H shows multilineage analysis of representative samples from
three different graft series, demonstrating lineage variability between individuals.

Single-cell transcriptome and functional analysis of the grafted mouse BM

To get an unbiased view of the human cells in the mice BM after engraftment, we
performed 10X Genomics on the BM of two representative primary mice (L5 and L1) 20 weeks
after transplantation. As shown in Figure 3A-C and in Figure S2A, the BM of both recipients
contained human erythroid, B, T, plasmacytoid dendritic, monocytic, and MAST cells (the
strategy for distinguishing human and mouse cells is described in detail in the Materials and
Methods section). The proportion of the different cell types was consistent between scRNA-
Seq and FACS analysis and between the two mice (Figures 3D-F and Figure S2A-D). Human
erythroid cells expressed E-, D-, F-, and G-globins, indicating mature erythropoiesis (Figure 3G).

To substantiate the functionality of the transplanted cells, we performed a human-
selective clonogenic assay using BM cells isolated from primary recipients. We obtained an
overall frequency of 17.5±2.4 colonies from 10⁴ BM cells (Figure 4A), (to be compared with
19.2±3.8 colonies from 10⁴ total BM cells obtained for the CD34 primary recipients). These
colonies were divided into CFU-GEMM (Figure 4A and 4B1), BFU-E (Figure 4B2), and CFU-GM
colonies (Figure 4B3). Cytological analysis of colonies revealed the presence of mature
macrophages (Figure 4C1), histiomonocytes (Figure 4C2), and erythroblasts (Figure 4C3). We
also analyzed the ability of human erythroid progenitor cells isolated from mouse BM to
undergo hemoglobin switching in vivo (n=3, 30 recipients). RT-qPCR analysis of total BM cells and erythroid colonies in vitro revealed high levels of human adult β- (39.51±4.95% and 28.91±2.43%, respectively) and foetal γ-(57.49±3.95% and 68.70±5.55%, respectively) globin gene expression, whereas embryonic ε-globin was dramatically reduced to 3.0±1.2% and 2.39±1.59% of total globin content, respectively (Figure 3D), consistent with the scRNA-seq analysis (Figure 3G). This is a hallmark of definitive erythropoiesis in the primary recipients and comparable to the globin content obtained during erythroid differentiation of CD34+ CB cells in vitro (Figure 4D), except for a small amount of epsilon globin, which is present in colonies derived from BM cells. Similar results were found when we analyzed globin content of individual BFU-E from primary recipients transplanted with D17 EB cells (n=24) and BFU-E from CD34+ CB cells (n=24) (Figure S3A).

As for the T lineage, BM had a significant proportion of hCD3+ cells (Figure 2F), similar to the content of hCD3+ cells found in the thymus (Figure 2G). In addition, thymus of the recipient mice contained significant amounts of hCD4+ and hCD8+ cells, together with TCRαβ expression combined to a low percentage of TCRγδ, the distribution of which being reminiscent of normal T lymphopoiesis (Figure 2G). We used a physiological method that can efficiently test T cell stimulation and proliferation <sup>49</sup>. The thymic hCD3 T cell population was CFSE labeled and stimulated by hCD3 and hCD28 (n=3, 30 thymuses). Cell numbers increased over the course of 5 days (Figure 4F) thereby demonstrating T cell activation and expansion. The presence of B and T cells was also confirmed by q-PCR of CD19 (B lymphocyte surface antigen B4) and TCF7 (Transcription Factor 7), respectively, in BM, thymus, and spleen (Figure S3B).

A hematopoietic progenitor cell assay revealed the presence of granulocytes and erythroblasts (Figure S3C). A TaqMan® RT-qPCR assay using the Human Hematopoiesis Array disclosed the presence of the canonical blood cell markers in the CD45+ hematopoietic fraction of BM (Figure S3D). To estimate the number of functionally engrafting human HSCs, we performed a limiting dilution assay <sup>50</sup> in primary NSG recipients and estimated that one D17 cell out of 15,700 is a fully SCID-repopulating cell (Figure 3G, Table S4).

To confirm the presence of human self-renewing HSCs, secondary transplants (n=40, Table S3) were performed. For each primary recipient, one secondary recipient was engrafted
undergo hemoglobin switching *in vivo* (n=3, 30 recipients). RT-qPCR analysis of total BM cells and erythroid colonies *in vitro* revealed high levels of human adult β- (39.51±4.95% and 28.91±2.43%, respectively) and foetal γ-(57.49±3.95% and 68.70±5.55%, respectively) globin gene expression, whereas embryonic ε-globin was dramatically reduced to 3.0±1.2% and 2.39±1.59% of total globin content, respectively (Figure 3D), consistent with the scRNA-seq analysis (Figure 3G). This is a hallmark of definitive erythropoiesis in the primary recipients and comparable to the globin content obtained during erythroid differentiation of CD34⁺ CB cells *in vitro* (Figure 4D), except for a small amount of epsilon globin, which is present in colonies derived from BM cells. Similar results were found when we analyzed globin content of individual BFU-E from primary recipients transplanted with D17 EB cells (n=24) and BFU-E from CD34⁺ CB cells (n=24) (Figure S3A).

As for the T lineage, BM had a significant proportion of hCD3⁺ cells (Figure 2F), similar to the content of hCD3⁺ cells found in the thymus (Figure 2G). In addition, thymus of the recipient mice contained significant amounts of hCD4⁺ and hCD8⁺ cells, together with TCRαβ expression combined to a low percentage of TCRγδ, the distribution of which being reminiscent of normal T lymphopoiesis (Figure 2G). We used a physiological method that can efficiently test T cell stimulation and proliferation <sup>49</sup>. The thymic hCD3 T cell population was CFSE labeled and stimulated by hCD3 and hCD28 (n=3, 30 thymuses). Cell numbers increased over the course of 5 days (Figure 4F) thereby demonstrating T cell activation and expansion. The presence of B and T cells was also confirmed by q-PCR of CD19 (B lymphocyte surface antigen B4) and TCF7 (Transcription Factor 7), respectively, in BM, thymus, and spleen (Figure S3B).

A hematopoietic progenitor cell assay revealed the presence of granulocytes and erythroblasts (Figure S3C). A TaqMan® RT-qPCR assay using the Human Hematopoiesis Array disclosed the presence of the canonical blood cell markers in the CD45⁺ hematopoietic fraction of BM (Figure S3D). To estimate the number of functionally engrafting human HSCs, we performed a limiting dilution assay <sup>50</sup> in primary NSG recipients and estimated that one D17 cell out of 15,700 is a fully SCID-repopulating cell (Figure 3G, Table S4).

To confirm the presence of human self-renewing HSCs, secondary transplants (n=40, Table S3) were performed. For each primary recipient, one secondary recipient was engrafted
with half of the flushed BM corresponding to $7.10^6$ BM cells (Figure 2A). Twenty weeks after transplantation, $hCD45^+$ cells accounted for $12.2\pm0.8\%$ of mononucleated BM cells (Figure 5A, representative sample), $8.1\pm0.6\%$ of $CD43^+$, and $4.8\pm0.6\%$ of $CD34^+$ cells (Figure 5B), demonstrating their sustained reconstitution capacity. In 40 of 40 secondary mice receiving BM cells, hematopoietic reconstitution was comparable to the pattern obtained with secondary $CD34^+$ CB cell transplantation and did not differ between individual hiPSC lines (Figure 5C, D). Figure 5E shows the reconstitution of the different HC lineages analyzed by flow cytometry in representative BM samples from secondary mice transplanted with primary mouse BM cells that received D17 EB cells, saline injection (Figure S4A) or $CD34^+$ CB cells (Figure S4B).

The cloning efficiency of human colony-forming cells was $7.1\pm2.0$ out of a total of $10^4$ BM cells from secondary recipients transplanted with EB cell progeny and $11.0\pm2.9$ out of a total of $10^4$ mouse cells BM derived from secondary recipients transplanted with $CD34^+$ cells, indicating a robust and long-lasting self-renewal potential (Figure 5F). The cells generated human erythroid progenitors with hallmarks of definitive erythropoiesis, i.e., high levels of $\beta$- (36.61$\pm$5.86%) and $\gamma$-globin (61.39$\pm$4.86%), whereas $\epsilon$-globin was drastically reduced to $2.1\pm1.1\%$ of total globins (Figure 4D), as analyzed by RT-qPCR.

A successful three-round serial-transplant assay was performed in NOD-SCID mice with $4.10^5$, $7.10^6$ and $7.10^6$ cells inoculated for primary, secondary and tertiary recipients respectively. All three tertiary-recipient mice exhibited multilineage engraftment at 12 weeks (Figure S4C, D, E), demonstrating that D17-EB-derived human HSCs display robust multilineage and self-renewal potential in vivo.

## Functional, molecular and phenotypic identification of the EB cells.

To better document our differentiation procedure, we transcriptionally profiled EBs at 4 different time points, i.e., D13, D15, D17, and D19 of culture, using 10X Genomics scRNA-Seq (n=2 biological replicates per time point for a total of 26,027 cells). UMAP visualization of the merged datasets identified 27 different cell types distributed among three main UMAP branches corresponding to the three germ layers, i.e., ectoderm, mesoderm and endoderm,
with half of the flushed BM corresponding to $7.10^6$ BM cells (Figure 2A). Twenty weeks after transplantation, $hCD45^+$ cells accounted for $12.2\pm0.8\%$ of mononucleated BM cells (Figure 5A, representative sample), $8.1\pm0.6\%$ of CD43$^+$, and $4.8\pm0.6\%$ of CD34$^+$ cells (Figure 5B), demonstrating their sustained reconstitution capacity. In 40 of 40 secondary mice receiving BM cells, hematopoietic reconstitution was comparable to the pattern obtained with secondary CD34$^+$ CB cell transplantation and did not differ between individual hiPSC lines (Figure 5C, D). Figure 5E shows the reconstitution of the different HC lineages analyzed by flow cytometry in representative BM samples from secondary mice transplanted with primary mouse BM cells that received D17 EB cells, saline injection (Figure S4A) or CD34$^+$ CB cells (Figure S4B).

The cloning efficiency of human colony-forming cells was $7.1\pm2.0$ out of a total of $10^4$ BM cells from secondary recipients transplanted with EB cell progeny and $11.0\pm2.9$ out of a total of $10^4$ mouse cells BM derived from secondary recipients transplanted with CD34$^+$ cells, indicating a robust and long-lasting self-renewal potential (Figure 5F). The cells generated human erythroid progenitors with hallmarks of definitive erythropoiesis, i.e., high levels of $\beta$- (36.61$\pm$5.86%) and $\gamma$-globin (61.39$\pm$4.86%), whereas $\epsilon$-globin was drastically reduced to $2.1\pm1.1\%$ of total globins (Figure 4D), as analyzed by RT-qPCR.

A successful three-round serial-transplant assay was performed in NOD-SCID mice with $4.10^5$, $7.10^6$ and $7.10^6$ cells inoculated for primary, secondary and tertiary recipients respectively. All three tertiary-recipient mice exhibited multilineage engraftment at 12 weeks (Figure S4C, D, E), demonstrating that D17-EB-derived human HSCs display robust multilineage and self-renewal potential in vivo.

## Functional, molecular and phenotypic identification of the EB cells.

To better document our differentiation procedure, we transcriptionally profiled EBs at 4 different time points, i.e., D13, D15, D17, and D19 of culture, using 10X Genomics scRNA-Seq (n=2 biological replicates per time point for a total of 26,027 cells). UMAP visualization of the merged datasets identified 27 different cell types distributed among three main UMAP branches corresponding to the three germ layers, i.e., ectoderm, mesoderm and endoderm,
and a fourth branch containing cells of the trophoblast lineage (Figure 6A and Figure S5). To better characterize the events leading to HSC development, we focused on the endothelial and HC clusters (Figure 6A, top left). As a first approach, we compared this cell subset with a published scRNA-Seq dataset from the human AGM region at Carnegie stage (CS) 16, the time of HSC development ⁵¹, (Figure 6B). Both data sets showed two types of vascular endothelial cells, an arterial-like CD34⁺, CDH5⁺, HEY2⁺, GJA5⁺ cluster and a venous-like CD34⁺, CDH5⁺, NR2F2⁺, APLNR⁺ cluster (Figure 6C, D). These data suggest that our differentiation procedure produces populations similar to those in the aorta of the human embryo when HSCs arise from the hemogenic endothelium.

To further explore this hypothesis, we examined the pattern of expression of MYB and RUNX1 by RT-qPCR and compared it with the profile obtained by scRNA-Seq (Figure 7A) from D13 to D17. An increase in their expression after D15 was observed by both methods. Flow cytometric analysis of endothelial markers (CD34, CDH5) showed a decrease in endothelial cells after D13 and the gradual appearance of the PTPRC⁺ population between D15 and 17 (Figure 7B). We also tested the endothelial and hematopoietic capacity of EB cells at D13, 17, and 19 by dedicated assays *in vitro* (Figure S6). D15 EB cells showed a strong endothelial potential, as evidenced by the formation of pseudo microtubules (Figure S6A) ⁵², and a poor hematopoietic capacity as they were able to form only a few clonogenic colonies and had a very low frequency of LTC-ICs (Figure S6B-D). In contrast, D17 cells lost endothelial potential but increased their hematopoietic potential (Figure S6A-D). Thus D17 EB cells have the highest HSC potential, coherent with the onset of hematopoiesis. Immunostaining of sections of D17 EBs showed that endothelial CDH5⁺CD31⁺ cells and NG2⁺ pericytes were distributed throughout the periphery of the EBs and absent from the center (Figure 7D, E, F, G), as assessed relative to the interior of this endothelium-rich outer region which is bordered by a ring of SMA⁺cells (Figure 7G). Interestingly, clusters of RUNX1⁺ endothelial and HCs were also identified (Figure 7F). This suggests that the periphery of EBs contains a dense vascular network in which there is likely a transition from endothelial cells to HCs, confirming the scRNA-Seq results.

Finally, we identified the subpopulations of HSCs produced. We projected the recently published six-gene signature and HSC score ⁵³, which distinguishes human embryonic HSCs from hematopoietic progenitor cells, onto the scRNA-Seq hematopoietic clusters (Figure 6E).
and a fourth branch containing cells of the trophoblast lineage (Figure 6A and Figure S5). To better characterize the events leading to HSC development, we focused on the endothelial and HC clusters (Figure 6A, top left). As a first approach, we compared this cell subset with a published scRNA-Seq dataset from the human AGM region at Carnegie stage (CS) 16, the time of HSC development ⁵¹, (Figure 6B). Both data sets showed two types of vascular endothelial cells, an arterial-like CD34⁺, CDH5⁺, HEY2⁺, GJA5⁺ cluster and a venous-like CD34⁺, CDH5⁺, NR2F2⁺, APLNR⁺ cluster (Figure 6C, D). These data suggest that our differentiation procedure produces populations similar to those in the aorta of the human embryo when HSCs arise from the hemogenic endothelium.

To further explore this hypothesis, we examined the pattern of expression of MYB and RUNX1 by RT-qPCR and compared it with the profile obtained by scRNA-Seq (Figure 7A) from D13 to D17. An increase in their expression after D15 was observed by both methods. Flow cytometric analysis of endothelial markers (CD34, CDH5) showed a decrease in endothelial cells after D13 and the gradual appearance of the PTPRC⁺ population between D15 and 17 (Figure 7B). We also tested the endothelial and hematopoietic capacity of EB cells at D13, 17, and 19 by dedicated assays *in vitro* (Figure S6). D15 EB cells showed a strong endothelial potential, as evidenced by the formation of pseudo microtubules (Figure S6A) ⁵², and a poor hematopoietic capacity as they were able to form only a few clonogenic colonies and had a very low frequency of LTC-ICs (Figure S6B-D). In contrast, D17 cells lost endothelial potential but increased their hematopoietic potential (Figure S6A-D). Thus D17 EB cells have the highest HSC potential, coherent with the onset of hematopoiesis. Immunostaining of sections of D17 EBs showed that endothelial CDH5⁺CD31⁺ cells and NG2⁺ pericytes were distributed throughout the periphery of the EBs and absent from the center (Figure 7D, E, F, G), as assessed relative to the interior of this endothelium-rich outer region which is bordered by a ring of SMA⁺cells (Figure 7G). Interestingly, clusters of RUNX1⁺ endothelial and HCs were also identified (Figure 7F). This suggests that the periphery of EBs contains a dense vascular network in which there is likely a transition from endothelial cells to HCs, confirming the scRNA-Seq results.

Finally, we identified the subpopulations of HSCs produced. We projected the recently published six-gene signature and HSC score ⁵³, which distinguishes human embryonic HSCs from hematopoietic progenitor cells, onto the scRNA-Seq hematopoietic clusters (Figure 6E).
Several RUNX1⁺ HOXA9⁺ MLLT3⁺ MECOM⁺ HLF⁺ SPINK2⁺ cells clustered into a candidate HSC subpopulation (Figure 6F). Refined comparisons with additional markers of human HSC ontogeny suggest that these candidate HSCs exhibit an intermediate phenotype between CS10 early AGM HSCs⁵⁴ and CS14-15 HSCs⁵³, with some early markers such as LIN28, GAD1, and FGF23 maintained alongside more mature markers such as HOXA9, PTPRC, and STAT5A (Table S8). Overall, the differentiation protocol presented here and applied to EBs appears to recapitulate human AGM formation and to give rise to HSCs with an early to intermediate AGM phenotype.

DISCUSSION

We successfully produced HSCs capable of long-term transplantation and self-renewal from hiPSCs using a combination of cytokines and growth factors, defined by DOE.
Several RUNX1⁺ HOXA9⁺ MLLT3⁺ MECOM⁺ HLF⁺ SPINK2⁺ cells clustered into a candidate HSC subpopulation (Figure 6F). Refined comparisons with additional markers of human HSC ontogeny suggest that these candidate HSCs exhibit an intermediate phenotype between CS10 early AGM HSCs⁵⁴ and CS14-15 HSCs⁵³, with some early markers such as LIN28, GAD1, and FGF23 maintained alongside more mature markers such as HOXA9, PTPRC, and STAT5A (Table S8). Overall, the differentiation protocol presented here and applied to EBs appears to recapitulate human AGM formation and to give rise to HSCs with an early to intermediate AGM phenotype.

DISCUSSION

We successfully produced HSCs capable of long-term transplantation and self-renewal from hiPSCs using a combination of cytokines and growth factors, defined by DOE.
Importantly, this was achieved by a one-step, GMP-compliant, procedure, without the use of reprogramming vectors.

We used DOE to finely tune a combination and concentrations of cytokines and growth factors that enabled the formation of HSCs. The protocol is extremely robust, as HSCs with similar reconstitution potential, approaching the reconstitution potential of CD34+ CB cells, were from five different hiPSC lines, regardless of the reprogramming method. A scaling-up of the protocol was successfully implemented since GMP-compliant batches of D17 cells have been produced with equal efficiency in an advanced therapy medicinal product platform (Atlantic Bio GMP Platform, Nantes; France). In conclusion, both the robustness and the reproducibility of this protocol meet the requirements of clinical applications.

According to scRNA-Seq data, we generate about 4.6% HCs at D17 and around 1 in 10,000 cells carries the six-gene signature of embryonic HSCs <sup>53</sup>. This is fully consistent with our limiting dilution experiments showing that 1 in 15,700 cells is an HSC. This highly efficient engraftment is in line with data showing that, although each human AGM contains only 1 or 2 HSCs, they exhibit a high repopulation capacity <sup>55</sup>. Similarly, our hiPSC-derived HSCs show a very robust reconstitution potential in multiple recipients with the full spectrum of hematopoietic lineages in BM, circulating blood, thymus, and spleen. In addition, no signs of leukemia or teratomas were observed in any of the transplanted mice.

The main issue with *de novo* generation of HSCs is the clonal reconstitution and the presence of multipotent progenitor cells instead of true HSCs. The latter is of particular concern because NSG mice are known to exhibit a B lymphoid bias <sup>56-58</sup>. Primary recipients show variable grafting patterns, both in terms of hCD45 expression and composition of the different HC lineages, and the secondary recipients show grafting patterns distinct from their primary counterparts. This argues against clonal reconstitution. In addition, individual BFU-E colonies performed from a single transplanted mouse BM showed different ratios of β-, γ-, and ε-globins. This suggests that erythropoiesis is maintained by multiple progenitor cells and not by a clonal mechanism.

ScRNA-Seq analysis of the D17 EBS reveals 2 major endothelial clusters: an arterial-like GJA5+ cluster and a venous-like APLNR+ cluster. These profiles are essentially the same as those described for the human CS16 embryo <sup>51</sup>. Interestingly, Calvanese and colleagues <sup>53</sup>
Importantly, this was achieved by a one-step, GMP-compliant, procedure, without the use of reprogramming vectors.

We used DOE to finely tune a combination and concentrations of cytokines and growth factors that enabled the formation of HSCs. The protocol is extremely robust, as HSCs with similar reconstitution potential, approaching the reconstitution potential of CD34+ CB cells, were from five different hiPSC lines, regardless of the reprogramming method. A scaling-up of the protocol was successfully implemented since GMP-compliant batches of D17 cells have been produced with equal efficiency in an advanced therapy medicinal product platform (Atlantic Bio GMP Platform, Nantes; France). In conclusion, both the robustness and the reproducibility of this protocol meet the requirements of clinical applications.

According to scRNA-Seq data, we generate about 4.6% HCs at D17 and around 1 in 10,000 cells carries the six-gene signature of embryonic HSCs <sup>53</sup>. This is fully consistent with our limiting dilution experiments showing that 1 in 15,700 cells is an HSC. This highly efficient engraftment is in line with data showing that, although each human AGM contains only 1 or 2 HSCs, they exhibit a high repopulation capacity <sup>55</sup>. Similarly, our hiPSC-derived HSCs show a very robust reconstitution potential in multiple recipients with the full spectrum of hematopoietic lineages in BM, circulating blood, thymus, and spleen. In addition, no signs of leukemia or teratomas were observed in any of the transplanted mice.

The main issue with *de novo* generation of HSCs is the clonal reconstitution and the presence of multipotent progenitor cells instead of true HSCs. The latter is of particular concern because NSG mice are known to exhibit a B lymphoid bias <sup>56-58</sup>. Primary recipients show variable grafting patterns, both in terms of hCD45 expression and composition of the different HC lineages, and the secondary recipients show grafting patterns distinct from their primary counterparts. This argues against clonal reconstitution. In addition, individual BFU-E colonies performed from a single transplanted mouse BM showed different ratios of β-, γ-, and ε-globins. This suggests that erythropoiesis is maintained by multiple progenitor cells and not by a clonal mechanism.

ScRNA-Seq analysis of the D17 EBS reveals 2 major endothelial clusters: an arterial-like GJA5+ cluster and a venous-like APLNR+ cluster. These profiles are essentially the same as those described for the human CS16 embryo <sup>51</sup>. Interestingly, Calvanese and colleagues <sup>53</sup>
describe the hemogenic endothelium as GJA5+ in the aorta of the human CS14 embryo. In
contrast, some cells of the APLNR+ endothelial cluster colocalize with the HCs cluster,
suggesting that the HSCs obtained are likely derived from an APLNR+ hemogenic endothelium.
This is consistent with other findings describing a possible switch in the identity of the
hemogenic endothelium during development from a venous to an arterial phenotype
between CS10 and CS13<sup>54</sup>. Since EB-derived HSCs identified, thanks to the six-gene embryonic
HSC signature<sup>53</sup>, exhibit an intermediate phenotype between CS10 and CS14, it seems highly
plausible that the hemogenic endothelium from which they arise also display an earlier
phenotype than CS14. Overall, our protocol likely recapitulates the emergence of HSCs from
the human AGM between CS10 and CS14.

Thus, the plasticity of the hemogenic endothelium phenotype argues for an important
role of the microenvironment in HSC production. This is reminiscent of the protocols that have
succeeded in generating functional HSCs from teratomas <sup>25 26</sup>. These protocols have
demonstrated that a variety of cell types from all germ layers can provide a rich
microenvironment <sup>27</sup> for generating HSCs suitable for long-term transplantation. Indeed, HSC
formation in the AGM is driven by signals from a variety of sources, including non-mesodermal
tissues such as the sympathetic nervous system <sup>59</sup>. The diversity of communications between
cell types in teratomas likely reflects these signals, which are sent from different tissues during
development and are critical for HSC to be produced. Similarly, EBs contain cells from the
ectoderm, endoderm, and mesoderm. This ensemble of cell types may form a rich, complex
microenvironment critical for the formation of transplantable HSCs. This may explain why our
protocol succeeds in generating HSCs with long-term transplantability, whereas most
protocols using EBs report cells with short-term potential.

**Limitation of the study**
describe the hemogenic endothelium as GJA5+ in the aorta of the human CS14 embryo. In
contrast, some cells of the APLNR+ endothelial cluster colocalize with the HCs cluster,
suggesting that the HSCs obtained are likely derived from an APLNR+ hemogenic endothelium.
This is consistent with other findings describing a possible switch in the identity of the
hemogenic endothelium during development from a venous to an arterial phenotype
between CS10 and CS13 <sup>54</sup>. Since EB-derived HSCs identified, thanks to the six-gene embryonic
HSC signature <sup>53</sup>, exhibit an intermediate phenotype between CS10 and CS14, it seems highly
plausible that the hemogenic endothelium from which they arise also display an earlier
phenotype than CS14. Overall, our protocol likely recapitulates the emergence of HSCs from
the human AGM between CS10 and CS14.

Thus, the plasticity of the hemogenic endothelium phenotype argues for an important
role of the microenvironment in HSC production. This is reminiscent of the protocols that have
succeeded in generating functional HSCs from teratomas <sup>25</sup> <sup>26</sup>. These protocols have
demonstrated that a variety of cell types from all germ layers can provide a rich
microenvironment <sup>27</sup> for generating HSCs suitable for long-term transplantation. Indeed, HSC
formation in the AGM is driven by signals from a variety of sources, including non-mesodermal
tissues such as the sympathetic nervous system <sup>59</sup>. The diversity of communications between
cell types in teratomas likely reflects these signals, which are sent from different tissues during
development and are critical for HSC to be produced. Similarly, EBs contain cells from the
ectoderm, endoderm, and mesoderm. This ensemble of cell types may form a rich, complex
microenvironment critical for the formation of transplantable HSCs. This may explain why our
protocol succeeds in generating HSCs with long-term transplantability, whereas most
protocols using EBs report cells with short-term potential.

**Limitation of the study**
EBs harbor an ensemble of cell types that may serve as a microenvironment. We do not yet know what type of tissue, cell type, and molecular interactions are required to promote HSC formation, but investigations are ongoing.

In the near future, it will be necessary to find markers for enrichment in endothelial/hematopoietic lineages if HSCs are to be generated for clinical translation. These approaches are currently under investigation.

Granulocytes and neutrophils are difficult to detect under our experimental conditions because samples are not processed immediately after collection and RNA content is low and RNases are relatively high, resulting in fewer transcripts detected and fewer usable sequencing reads using 10X technology.

We do not know whether the transplanted HSCs require non-hematopoietic tissue to develop in the mouse BM. A time course analysis of the transplanted non-hematopoietic tissues is underway.

Such a transgene- and feeder-free tool could pave the way for future clinical applications and the discovery of therapeutic targets by providing a model for deciphering the emergence of hereditary blood malignancies. Our system is also a real step towards a better understanding of developing hematopoiesis in the human embryo, as it can overcome the ethical constraints associated with human embryo research.
EBs harbor an ensemble of cell types that may serve as a microenvironment. We do not yet know what type of tissue, cell type, and molecular interactions are required to promote HSC formation, but investigations are ongoing.

In the near future, it will be necessary to find markers for enrichment in endothelial/hematopoietic lineages if HSCs are to be generated for clinical translation. These approaches are currently under investigation.

Granulocytes and neutrophils are difficult to detect under our experimental conditions because samples are not processed immediately after collection and RNA content is low and RNases are relatively high, resulting in fewer transcripts detected and fewer usable sequencing reads using 10X technology.

We do not know whether the transplanted HSCs require non-hematopoietic tissue to develop in the mouse BM. A time course analysis of the transplanted non-hematopoietic tissues is underway.

Such a transgene- and feeder-free tool could pave the way for future clinical applications and the discovery of therapeutic targets by providing a model for deciphering the emergence of hereditary blood malignancies. Our system is also a real step towards a better understanding of developing hematopoiesis in the human embryo, as it can overcome the ethical constraints associated with human embryo research.
## AUTHOR CONTRIBUTIONS

L.G.H., OP, and MBM performed the experiments with assistance from LP, BL, BB, and CL. OP performed the single-cell transcriptome analysis. TZ and LM replicated the culture protocol independently in an ATMP platform. LP replicated the FACS analysis independently. H.L., L.D., and A.C. helped with the design of the study. M.B. helped design the animal study. A.C., T.J., and L.G.H. monitored the study. L.G.H. and T.J. designed the study, analyzed the data, interpreted the experiments, and wrote the manuscript.

## ACKNOWLEDGMENTS

We thank S. Viville (IGBMC, Strasbourg, France) and L. David (ISPCDC, Nantes, France) for providing iPS lines, Rima Haddad for scientific support and Sophie Gournet for excellent drawing assistance. This work benefited from equipment and services from the iGenSeq core facility, at ICM. We warmly thank F. Letourneur (Plateforme génomique Institut Cochin Paris) and to Y. Marie (Plateforme Génomique ICM) for help in single-cell sequencing and demultiplexing. Part of the bioinformatics analyses was performed on the Core Cluster of the Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013). We are indebted to the Fondation Ramsay Santé and Assistance Publique-Hôpitaux de Paris for providing the cord blood cells. This research was funded by the ANR-13-ASTR-009 "GIPSI" and ANR-19-ASMA-0005 "RESCUE" from the Direction Générale de l'Armement via the ASTRID/ANR program for AC, the ANR-20-CE17-0032-01 "HEMPLUS", the FRM grant EQU201903007867 for TJ and the Etablissement Français du Sang via the APR 2013 for LGH.
## AUTHOR CONTRIBUTIONS

L.G.H., OP, and MBM performed the experiments with assistance from LP, BL, BB, and CL. OP performed the single-cell transcriptome analysis. TZ and LM replicated the culture protocol independently in an ATMP platform. LP replicated the FACS analysis independently. H.L., L.D., and A.C. helped with the design of the study. M.B. helped design the animal study. A.C., T.J., and L.G.H. monitored the study. L.G.H. and T.J. designed the study, analyzed the data, interpreted the experiments, and wrote the manuscript.

## ACKNOWLEDGMENTS

We thank S. Viville (IGBMC, Strasbourg, France) and L. David (ISPCDC, Nantes, France) for providing iPS lines, Rima Haddad for scientific support and Sophie Gournet for excellent drawing assistance. This work benefited from equipment and services from the iGenSeq core facility, at ICM. We warmly thank F. Letourneur (Plateforme génomique Institut Cochin Paris) and to Y. Marie (Plateforme Génomique ICM) for help in single-cell sequencing and demultiplexing. Part of the bioinformatics analyses was performed on the Core Cluster of the Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013). We are indebted to the Fondation Ramsay Santé and Assistance Publique-Hôpitaux de Paris for providing the cord blood cells. This research was funded by the ANR-13-ASTR-009 "GIPSI" and ANR-19-ASMA-0005 "RESCUE" from the Direction Générale de l'Armement via the ASTRID/ANR program for AC, the ANR-20-CE17-0032-01 "HEMPLUS", the FRM grant EQU201903007867 for TJ and the Etablissement Français du Sang via the APR 2013 for LGH.
A CC-BY public copyright license has been applied by the authors to the present document and
will be applied to all subsequent versions up to the Author Accepted Manuscript arising from
this submission, in accordance with the grant's open access conditions.

**Declaration of interest**

The authors declare no competing interest

**Inclusion and diversity**

We support inclusive, diverse, and equitable conduct of research.
A CC-BY public copyright license has been applied by the authors to the present document and
will be applied to all subsequent versions up to the Author Accepted Manuscript arising from
this submission, in accordance with the grant's open access conditions.

**Declaration of interest**

The authors declare no competing interest

**Inclusion and diversity**

We support inclusive, diverse, and equitable conduct of research.
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2. Lapillonne, H., Kobari, L., Mazurier, C., Tropel, P., Giarratana, M.C., Zanella-Cleon, I., Kiger, L., Wattenhofer-Donze, M., Puccio, H., Hebert, N., et al. (2010). Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 95, 1651-1659. 10.3324/haematol.2010.023556.

3. Hirose, S., Takayama, N., Nakamura, S., Nagasawa, K., Ochi, K., Hirata, S., Yamazaki, S., Yamaguchi, T., Otsu, M., Sano, S., et al. (2013). Immortalization of erythroblasts by c-MYC and BCL-XL enables large-scale erythrocyte production from human pluripotent stem cells. Stem Cell Reports 1, 499-508. 10.1016/j.stemcr.2013.10.010.

4. Kurita, R., Suda, N., Sudo, K., Miharada, K., Hiroyama, T., Miyoshi, H., Tani, K., and Nakamura, Y. (2013). Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS One 8, e59890. 10.1371/journal.pone.0059890.

5. Carpenter, L., Malladi, R., Yang, C.T., French, A., Pilkington, K.J., Forsey, R.W., Sloane-Stanley, J., Silk, K.M., Davies, T.J., Fairchild, P.J., et al. (2011). Human induced pluripotent stem cells are capable of B-cell lymphopoiesis. Blood 117, 4008-4011. 10.1182/blood-2010-08-299941.

6. French, A., Yang, C.T., Taylor, S., Watt, S.M., and Carpenter, L. (2015). Human induced pluripotent stem cell-derived B lymphocytes express slgM and can be generated via a hemogenic endothelium intermediate. Stem Cells Dev 24, 1082-1095. 10.1089/scd.2014.0318.

7. Galic, Z., Kitchen, S.G., Kacena, A., Subramanian, A., Burke, B., Cortado, R., and Zack, J.A. (2006). T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A 103, 11742-11747. 10.1073/pnas.0604244103.

8. Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama-Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., et al. (2013). Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 12, 114-126. 10.1016/j.stem.2012.11.002.

9. Themeli, M., Kloss, C.C., Ciriello, G., Fedorov, V.D., Perna, F., Gonen, M., and Sadelain, M. (2013). Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol 31, 928-933. 10.1038/nbt.2678.

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11. Haruta, M., Tomita, Y., Yuno, A., Matsumura, K., Ikeda, T., Takamatsu, K., Haga, E., Koba, C., Nishimura, Y., and Senju, S. (2013). TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells. Gene Ther 20, 504-513. 10.1038/gt.2012.59.

12. Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., and Nakauchi, H. (2008). Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood 111, 5298-5306. 10.1182/blood-2007-10-117622.

13. Nakamura, S., Sugimoto, N., and Eto, K. (2021). Development of platelet replacement therapy using human induced pluripotent stem cells. Dev Growth Differ 63, 178-186. 10.1111/dgd.12711.
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16. Narayan, A.D., Chase, J.L., Lewis, R.L., Tian, X., Kaufman, D.S., Thomson, J.A., and Zanjani, E.D. (2006). Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients. Blood 107, 2180-2183. 10.1182/blood-2005-05-1922.

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19. Sandler, V.M., Lailler, N., and Bouhassira, E.E. (2011). Reprogramming of embryonic human fibroblasts into fetal hematopoietic progenitors by fusion with human fetal liver CD34+ cells. PLoS One 6, e18265. 10.1371/journal.pone.0018265.

20. Szabo, E., Rampalli, S., Risueno, R.M., Schnerch, A., Mitchell, R., Fiebig-Comyn, A., Levadoux-Martin, M., and Bhatia, M. (2010). Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 468, 521-526. 10.1038/nature09591.

21. Sandler, V.M., Lis, R., Liu, Y., Kedem, A., James, D., Elemento, O., Butler, J.M., Scandura, J.M., and Rafii, S. (2014). Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 312-318. 10.1038/nature13547.

22. Doulatov, S., Vo, L.T., Chou, S.S., Kim, P.G., Arora, N., Li, H., Hadland, B.K., Bernstein, I.D., Collins, J.J., Zon, L.I., and Daley, G.Q. (2013). Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell Stem Cell 13, 459-470. 10.1016/j.stem.2013.09.002.

23. Sugimura, R., Jha, D.K., Han, A., Soria-Valles, C., da Rocha, E.L., Lu, Y.F., Goettel, J.A., Serrao, E., Rowe, R.G., Malleshaiah, M., et al. (2017). Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432-438. 10.1038/nature22370.

24. Lis, R., Karrasch, C.C., Poulos, M.G., Kunar, B., Redmond, D., Duran, J.G.B., Badwe, C.R., Schachterle, W., Ginsberg, M., Xiang, J., et al. (2017). Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature 545, 439-445. 10.1038/nature22326.

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26. Suzuki, N., Yamazaki, S., Yamaguchi, T., Okabe, M., Masaki, H., Takaki, S., Otsu, M., and Nakauchi, H. (2013). Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther 21, 1424-1431. 10.1038/mt.2013.71.

27. McDonald, D., Wu, Y., Dailamy, A., Tat, J., Parekh, U., Zhao, D., Hu, M., Tipps, A., Zhang, K., and Mali, P. (2020). Defining the Teratoma as a Model for Multi-lineage Human Development. Cell 183, 1402-1419 e1418. 10.1016/j.cell.2020.10.018.

28. Dzierzak, E., and Bigas, A. (2018). Blood Development: Hematopoietic Stem Cell Dependence and Independence. Cell Stem Cell 22, 639-651. 10.1016/j.stem.2018.04.015.

29. Canu, G., and Ruhrberg, C. (2021). First blood: the endothelial origins of hematopoietic progenitors. Angiogenesis 24, 199-211. 10.1007/s10456-021-09783-9.
14. Flippe, L., Gaignerie, A., Serazin, C., Baron, O., Saulquin, X., Anegon, I., David, L., and Guillonneau, C. (2022). Generation of CD34(+)CD43(+) Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells. Cells **11**. 10.3390/cells11244046.

15. Wang, Y., Yates, F., Naveiras, O., Ernst, P., and Daley, G.Q. (2005). Embryonic stem cell-derived hematopoietic stem cells. Proc Natl Acad Sci U S A **102**, 19081-19086.

16. Narayan, A.D., Chase, J.L., Lewis, R.L., Tian, X., Kaufman, D.S., Thomson, J.A., and Zanjani, E.D. (2006). Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients. Blood **107**, 2180-2183. 10.1182/blood-2005-05-1922.

17. Ledran, M.H., Krassowska, A., Armstrong, L., Dimmick, I., Renstrom, J., Lang, R., Yung, S., Santibanez-Coref, M., Dzierzak, E., Stojkovic, M., et al. (2008). Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell **3**, 85-98.

18. Risueno, R.M., Sachlos, E., Lee, J.H., Lee, J.B., Hong, S.H., Szabo, E., and Bhatia, M. (2012). Inability of human induced pluripotent stem cell-hematopoietic derivatives to downregulate microRNAs in vivo reveals a block in xenograft hematopoietic regeneration. Stem Cells **30**, 131-139. 10.1002/stem.1684.

19. Sandler, V.M., Lailler, N., and Bouhassira, E.E. (2011). Reprogramming of embryonic human fibroblasts into fetal hematopoietic progenitors by fusion with human fetal liver CD34+ cells. PLoS One **6**, e18265. 10.1371/journal.pone.0018265.

20. Szabo, E., Rampalli, S., Risueno, R.M., Schnerch, A., Mitchell, R., Fiebig-Comyn, A., Levadoux-Martin, M., and Bhatia, M. (2010). Direct conversion of human fibroblasts to multilineage blood progenitors. Nature **468**, 521-526. 10.1038/nature09591.

21. Sandler, V.M., Lis, R., Liu, Y., Kedem, A., James, D., Elemento, O., Butler, J.M., Scandura, J.M., and Rafii, S. (2014). Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature **511**, 312-318. 10.1038/nature13547.

22. Doulatov, S., Vo, L.T., Chou, S.S., Kim, P.G., Arora, N., Li, H., Hadland, B.K., Bernstein, I.D., Collins, J.J., Zon, L.I., and Daley, G.Q. (2013). Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell Stem Cell **13**, 459-470. 10.1016/j.stem.2013.09.002.

23. Sugimura, R., Jha, D.K., Han, A., Soria-Valles, C., da Rocha, E.L., Lu, Y.F., Goettel, J.A., Serrao, E., Rowe, R.G., Malleshaiah, M., et al. (2017). Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature **545**, 432-438. 10.1038/nature22370.

24. Lis, R., Karrasch, C.C., Poulos, M.G., Kunar, B., Redmond, D., Duran, J.G.B., Badwe, C.R., Schachterle, W., Ginsberg, M., Xiang, J., et al. (2017). Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature **545**, 439-445. 10.1038/nature22326.

25. Amabile, G., Welner, R.S., Nombela-Arrieta, C., D'Alise, A.M., Di Ruscio, A., Ebralidze, A.K., Kraytsberg, Y., Ye, M., Kocher, O., Neuberg, D.S., et al. (2013). In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood **121**, 1255-1264. 10.1182/blood-2012-06-434407.

26. Suzuki, N., Yamazaki, S., Yamaguchi, T., Okabe, M., Masaki, H., Takaki, S., Otsu, M., and Nakauchi, H. (2013). Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther **21**, 1424-1431. 10.1038/mt.2013.71.

27. McDonald, D., Wu, Y., Dailamy, A., Tat, J., Parekh, U., Zhao, D., Hu, M., Tipps, A., Zhang, K., and Mali, P. (2020). Defining the Teratoma as a Model for Multi-lineage Human Development. Cell **183**, 1402-1419 e1418. 10.1016/j.cell.2020.10.018.

28. Dzierzak, E., and Bigas, A. (2018). Blood Development: Hematopoietic Stem Cell Dependence and Independence. Cell Stem Cell **22**, 639-651. 10.1016/j.stem.2018.04.015.

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32. Sturgeon, C.M., Ditadi, A., Clarke, R.L., and Keller, G. (2013). Defining the path to hematopoietic stem cells. *Nat Biotechnol* 31, 416-418. 10.1038/nbt.2571.

33. Slukvin, I. (2013). Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells. *Blood* 122, 4035-4046. 10.1182/blood-2013-07-474825.

34. Vo, L.T., and Daley, G.Q. (2015). De novo generation of HSCs from somatic and pluripotent stem cell sources. *Blood* 125, 2641-2648. 10.1182/blood-2014-10-570234.

35. Arboretti, R., Ceccato, R., Pegoraro, L., Salmaso, L., Housmekerides, C., Spadoni, L., Pierangelo, E., Quaggia, S., Tveit, C., and Vianello, S. (2022). Machine learning and design of experiments with an application to product innovation in the chemical industry. *J Appl Stat* 49, 2674-2699. 10.1080/02664763.2021.1907840.

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37. Chadwick, K., Wang, L., Li, L., Menendez, P., Murdoch, B., Rouleau, A., and Bhatia, M. (2003). Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. *Blood* 102, 906-915.

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53. Calvanese, V., Capellera-Garcia, S., Ma, F., Fares, I., Liebscher, S., Ng, E.S., Ekstrand, S., Aguade-Gorgorio, J., Vavilina, A., Lefaudeux, D., et al. (2022). Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature. 10.1038/s41586-022-04571-x.

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55. Ivanovs, A., Rybtsov, S., Welch, L., Anderson, R.A., Turner, M.L., and Medvinsky, A. (2011). Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region. J Exp Med 208, 2417-2427. jem.20111688 [pii] 10.1084/jem.20111688.

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60. Kobari, L., Pflumio, F., Giarratana, M., Li, X., Titeux, M., Izac, B., Leteurtre, F., Coulombel, L., and Douay, L. (2000). In vitro and in vivo evidence for the long-term multilineage (myeloid, B,
47. Audige, A., Rochat, M.A., Li, D., Ivic, S., Fahrny, A., Muller, C.K.S., Gers-Huber, G., Myburgh, R., Bredl, S., Schlaepfer, E., et al. (2017). Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells. BMC Immunol 18, 28. 10.1186/s12865-017-0209-9.

48. Shultz, L.D., Lyons, B.L., Burzenski, L.M., Gott, B., Chen, X., Chaleff, S., Kotb, M., Gillies, S.D., King, M., Mangada, J., et al. (2005). Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174, 6477-6489. 10.4049/jimmunol.174.10.6477.

49. Fulcher, D., and Wong, S. (1999). Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory. Immunol Cell Biol 77, 559-564. 10.1046/j.1440-1711.1999.00870.x.

50. Huang, X., Lee, M.R., Cooper, S., Hangoc, G., Hong, K.S., Chung, H.M., and Broxmeyer, H.E. (2016). Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression. Leukemia 30, 144-153. 10.1038/leu.2015.189.

51. Crosse, E.I., Gordon-Keylock, S., Rybtsov, S., Binagui-Casas, A., Felchle, H., Nnadi, N.C., Kirschner, K., Chandra, T., Tamagno, S., Webb, D.J., et al. (2020). Multi-layered Spatial Transcriptomics Identify Secretory Factors Promoting Human Hematopoietic Stem Cell Development. Cell Stem Cell 27, 822-839 e828. 10.1016/j.stem.2020.08.004.

52. Smadja, D.M., Cornet, A., Emmerich, J., Aiach, M., and Gaussem, P. (2007). Endothelial progenitor cells: characterization, in vitro expansion, and prospects for autologous cell therapy. Cell Biol Toxicol 23, 223-239. 10.1007/s10565-007-0177-6.

53. Calvanese, V., Capellera-Garcia, S., Ma, F., Fares, I., Liebscher, S., Ng, E.S., Ekstrand, S., Aguade-Gorgorio, J., Vavilina, A., Lefaudeux, D., et al. (2022). Mapping human haematopoietic stem cells from haemogenic endothelium to birth. Nature. 10.1038/s41586-022-04571-x.

54. Zeng, Y., He, J., Bai, Z., Li, Z., Gong, Y., Liu, C., Ni, Y., Du, J., Ma, C., Bian, L., et al. (2019). Tracing the first hematopoietic stem cell generation in human embryo by single-cell RNA sequencing. Cell Res 29, 881-894. 10.1038/s41422-019-0228-6.

55. Ivanovs, A., Rybtsov, S., Welch, L., Anderson, R.A., Turner, M.L., and Medvinsky, A. (2011). Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region. J Exp Med 208, 2417-2427. jem.20111688 [pii] 10.1084/jem.20111688.

56. Rongvaux, A., Takizawa, H., Strowig, T., Willinger, T., Eynon, E.E., Flavell, R.A., and Manz, M.G. (2013). Human hemato-lymphoid system mice: current use and future potential for medicine. Annu Rev Immunol 31, 635-674. 10.1146/annurev-immunol-032712-095921.

57. Sippel, T.R., Radtke, S., Olsen, T.M., Kiem, H.P., and Rongvaux, A. (2019). Human hematopoietic stem cell maintenance and myeloid cell development in next-generation humanized mouse models. Blood Adv 3, 268-274. 10.1182/bloodadvances.2018023887.

58. Ito, M., Hiramatsu, H., Kobayashi, K., Suzue, K., Kawahata, M., Hioki, K., Ueyama, Y., Koyanagi, Y., Sugamura, K., Tsuji, K., et al. (2002). NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175-3182. 10.1182/blood-2001-12-0207.

59. Fitch, S.R., Kimber, G.M., Wilson, N.K., Parker, A., Mirshekar-Syahkal, B., Gottgens, B., Medvinsky, A., Dzierzak, E., and Ottersbach, K. (2012). Signaling from the sympathetic nervous system regulates hematopoietic stem cell emergence during embryogenesis. Cell Stem Cell 11, 554-566. S1934-5909(12)00417-1 [pii] 10.1016/j.stem.2012.07.002.

60. Kobari, L., Pflumio, F., Giarratana, M., Li, X., Titeux, M., Izac, B., Leteurtre, F., Coulombel, L., and Douay, L. (2000). In vitro and in vivo evidence for the long-term multilineage (myeloid, B,
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63. Han, X., Zhou, Z., Fei, L., Sun, H., Wang, R., Chen, Y., Chen, H., Wang, J., Tang, H., Ge, W., et al. (2020). Construction of a human cell landscape at single-cell level. Nature 581, 303-309. 10.1038/s41586-020-2157-4.

64 Hao, Y., Hao, S., Andersen-Nissen, E., Mauck, W.M., 3rd, Zheng, S., Butler, A., Lee, M.J., Wilk, A.J., Darby, C., Zager, M., et al. (2021). Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 e3529. 10.1016/j.cell.2021.04.048.
NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells. Exp Hematol 28, 1470-1480. 10.1016/s0301-472x(00)00557-9.

61. Hu, Y., and Smyth, G.K. (2009). ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347, 70-78. 10.1016/j.jim.2009.06.008.

62. Young, M.D., and Behjati, S. (2020). SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data. Gigascience 9. 10.1093/gigascience/giaa151.

63. Han, X., Zhou, Z., Fei, L., Sun, H., Wang, R., Chen, Y., Chen, H., Wang, J., Tang, H., Ge, W., et al. (2020). Construction of a human cell landscape at single-cell level. Nature 581, 303-309. 10.1038/s41586-020-2157-4.

64 Hao, Y., Hao, S., Andersen-Nissen, E., Mauck, W.M., 3rd, Zheng, S., Butler, A., Lee, M.J., Wilk, A.J., Darby, C., Zager, M., et al. (2021). Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 e3529. 10.1016/j.cell.2021.04.048.
FIGURE LEGENDS

**Figure 1: Design of Experiment step by step.**

Based on a literature review (see references in Table S1), an ensemble of cytokines and growth factors was identified (input variables) and a range of input concentrations was defined for each of them. Output variables such as cell surface markers (Table S2), cell proliferation, and LTC-IC frequency were defined. Based on these variables, 16 experimental designs were created that differed in their individual concentrations of growth factors and cytokines (Table S1). Baseline variable data from the 16 different experimental designs were collected. Based on these results, mathematical modeling of the interactions between the input and output variables was calculated. Three final combinations were proposed and tested, first with flow cytometry analysis on two different hiPSC lines (ie, FD136-25 and IMR90-16) (n=1 and n=1 independent experiments respectively) and second with mouse transplants using the FD136-25 line (n=15, 2 independent experiments). Statistical *data are presented as box plots and are mean ± SD or SEM according to the number of independent experiments. Wilcoxon Mann-Whitney test.*

See also supplemental experimental procedures and Tables S1, S2, and S3.

**Figure 2: In vivo primary engraftment of D17 EB cells in immunocompromised NSG mice.**

(A) hiPSCs (5 different cell lines) were differentiated in liquid culture supplemented with combination A of growth factors and cytokines for a total period of 17 days. EB or CD34⁺ CB cells (4.10⁵) were dissociated and inoculated into irradiated primary NSG recipients over a total period of 20 weeks. BM cells from primary recipients were collected and inoculated into secondary irradiated NSG recipients (7.10⁶ cells) over a further period of 20 weeks.

(B) Flow cytometric analysis of human and mouse CD45⁺ cell expression in BM of a representative primary recipient.

(C) Multilineage analysis by FACS on NSG mice BM, 20 weeks post-transplantation, on hCD34⁺hCD43⁺ and hCD45⁺ HSPCs into primary recipients. All mixed hiPSC lines. Each circle/square/triangle represents a single animal. **n=59, 14 independent experiments.**

(D) Multilineage analysis from the five individual hiPSC lines: FD136-25 (n= 10 mice, 3 independent experiments, red circles), PC1429 (n=10, 3 independent experiments, orange
**Figure 1: Design of Experiment step by step.**

Based on a literature review (see references in Table S1), an ensemble of cytokines and growth factors was identified (input variables) and a range of input concentrations was defined for each of them. Output variables such as cell surface markers (Table S2), cell proliferation, and LTC-IC frequency were defined. Based on these variables, 16 experimental designs were created that differed in their individual concentrations of growth factors and cytokines (Table S1). Baseline variable data from the 16 different experimental designs were collected. Based on these results, mathematical modeling of the interactions between the input and output variables was calculated. Three final combinations were proposed and tested, first with flow cytometry analysis on two different hiPSC lines (ie, FD136-25 and IMR90-16) (n=1 and n=1 independent experiments respectively) and second with mouse transplants using the FD136-25 line (n=15, 2 independent experiments). Statistical *data are presented as box plots and are mean ± SD or SEM according to the number of independent experiments. Wilcoxon Mann-Whitney test.*

See also supplemental experimental procedures and Tables S1, S2, and S3.

**Figure 2: In vivo primary engraftment of D17 EB cells in immunocompromised NSG mice.**

(A) hiPSCs (5 different cell lines) were differentiated in liquid culture supplemented with combination A of growth factors and cytokines for a total period of 17 days. EB or CD34⁺ CB cells (4.10⁵) were dissociated and inoculated into irradiated primary NSG recipients over a total period of 20 weeks. BM cells from primary recipients were collected and inoculated into secondary irradiated NSG recipients (7.10⁶ cells) over a further period of 20 weeks.

(B) Flow cytometric analysis of human and mouse CD45⁺ cell expression in BM of a representative primary recipient.

(C) Multilineage analysis by FACS on NSG mice BM, 20 weeks post-transplantation, on hCD34⁺hCD43⁺ and hCD45⁺ HSPCs into primary recipients. All mixed hiPSC lines. Each circle/square/triangle represents a single animal. **n=59, 14 independent experiments.**

(D) Multilineage analysis from the five individual hiPSC lines: FD136-25 (n= 10 mice, 3 independent experiments, red circles), PC1429 (n=10, 3 independent experiments, orange
squares), PCi-CAU (n= 26, 4 independent experiments, green triangles), LAM001.005 (n=6, 2 independent experiments, light blue lozenges) and LAM002.002 (n=7, 2 independent experiments, dark blue lozenges) and the CD34+ CB cells (n=13, 3 independent experiments, grey circles) in NSG primary recipients 20 weeks after transplantation of 4x10⁵ D17 EB or CD34+ CB cells. FACS results are expressed as % of hCD34+, hCD43+, and hCD45+. The results are relatively equivalent between the lines and close to the CD34 control. The dotted line indicates the limit of 0.1% of hCD45+ usually considered positive for human hematopoietic transplantation in NSG mice⁴³.

(E) Distribution of human myeloid, B-lymphoid, and T-lymphoid cells (hCD33+, hCD19+, and hCD3+, respectively) and hCD235a+ erythroid progenitors/precursors in the grafted BM 20 weeks after transplantation of each of the five hiPSCs lines compared with CD34+ CB cells. Same number of animals and presentation as in (D).

(F) Flow cytometric analysis of a representative animal for myeloid (hCD14/hCD15), lymphoid (hCD19/hIgM, SSC-A/hCD3) and pro-erythroid (hCD71/hCD235a) lineages in the grafted BM. Myeloid and lymphoid cells are analyzed from hCD45+ gated BM cells whereas hCD71/hCD235a (Pro-E) was analyzed on whole BM cells.

(G) Flow cytometric analysis of a representative animal for the distribution of hCD3, hCD4 and hCD8 antigens and of hTCRαβ and γδ in T lymphoid cells in a grafted thymus.

(H) Distribution of T, B, Pro-erythrocyte and monocyte lineages in representative individual primary recipients analyzed by flow cytometry. Mice 1-6, a-e and L1-L5 are from one independent experiment analyzed at one week interval i.e., a-e and L1-L5 were analyzed at the same time by two independent laboratories.

Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.

<caption>**Figure 3: Single cell transcriptome and phenotypic analysis of the grafted NSG BM.**</caption>

(A, B) UMAP embedding of a merged data set from two representative transplanted mice (mice L1 and L5, displayed in Figure 2H). BM data set colored by cell type (A) and by sample (B).

(C) UMAP embedding of a merged dataset of human cells in the two engrafted BM datasets colored by cell type.
squares), PCi-CAU (n= 26, 4 independent experiments, green triangles), LAM001.005 (n=6, 2 independent experiments, light blue lozenges) and LAM002.002 (n=7, 2 independent experiments, dark blue lozenges) and the CD34+ CB cells (n=13, 3 independent experiments, grey circles) in NSG primary recipients 20 weeks after transplantation of 4x10⁵ D17 EB or CD34+ CB cells. FACS results are expressed as % of hCD34+, hCD43+, and hCD45+. The results are relatively equivalent between the lines and close to the CD34 control. The dotted line indicates the limit of 0.1% of hCD45+ usually considered positive for human hematopoietic transplantation in NSG mice⁴³.

(E) Distribution of human myeloid, B-lymphoid, and T-lymphoid cells (hCD33+, hCD19+, and hCD3+, respectively) and hCD235a+ erythroid progenitors/precursors in the grafted BM 20 weeks after transplantation of each of the five hiPSCs lines compared with CD34+ CB cells. Same number of animals and presentation as in (D).

(F) Flow cytometric analysis of a representative animal for myeloid (hCD14/hCD15), lymphoid (hCD19/hIgM, SSC-A/hCD3) and pro-erythroid (hCD71/hCD235a) lineages in the grafted BM. Myeloid and lymphoid cells are analyzed from hCD45+ gated BM cells whereas hCD71/hCD235a (Pro-E) was analyzed on whole BM cells.

(G) Flow cytometric analysis of a representative animal for the distribution of hCD3, hCD4 and hCD8 antigens and of hTCRαβ and γδ in T lymphoid cells in a grafted thymus.

(H) Distribution of T, B, Pro-erythrocyte and monocyte lineages in representative individual primary recipients analyzed by flow cytometry. Mice 1-6, a-e and L1-L5 are from one independent experiment analyzed at one week interval i.e., a-e and L1-L5 were analyzed at the same time by two independent laboratories.

Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.

<caption>**Figure 3: Single cell transcriptome and phenotypic analysis of the grafted NSG BM.**</caption>

(A, B) UMAP embedding of a merged data set from two representative transplanted mice (mice L1 and L5, displayed in Figure 2H). BM data set colored by cell type (A) and by sample (B).

(C) UMAP embedding of a merged dataset of human cells in the two engrafted BM datasets colored by cell type.
(D) UMAP representation of single-cell RNA-Seq from NSG mouse L5 BM (14173 cells) transplanted with D17 EB cells for 20 weeks. Mouse and human cell clusters are identified.

(E-F) Bone marrow of NSG mouse L5 primary recipient analyzed by FACS at 20 weeks for (E) human erythroid cells (hCD235a⁺ and hCD235a+hCD71⁺) and (F) B cells (hIgM⁺CD10⁺), neutrophils (hCD33⁺hCD14⁺), and T cells (hCD4⁺hCD3⁺).

(G) Expression of globin genes in human early erythroid cells in transplanted mouse bone marrow datasets.

**Figure 4: Functional assessment of the grafted cells**

(A) Clonogenic hematopoietic assays on BM cells isolated from primary recipients grafted with either D17 EB (n=59, 14 independent experiments) or CD34⁺ CB cells (n=13, 3 independent experiments). Frequency of CFU-GM, BFU-E, and CFU-GEMM colonies.

(B) Representative colonies of CFU-GEMM (1), CFU-E (2) and CFU-GM (3) from D17 EB primary BM cells (n=59, 14 independent experiments).

(C) Cytospins. May Grünwald-Giemsa staining of cells isolated from clonogenic assays in primary or secondary recipients. Mature macrophages (1), histio monocytes (2), myelocytes (2), and erythroblasts (3). **B: bar=50µm; C: bar= 10µm**

(D) Human globin expression from CB CD34⁺HSPCs erythroid culture (n=3), from BM primary (n=12) and secondary recipients (n=4) and pooled BFU-E from BM primary recipients (6 to 10 BFU-E pooled. n=12, 3 independent experiments. Data are mean ± SEM

(E) Maturation of human T cells. Peripheral blood from a representative grafted primary recipient stained with antibodies against hTCR αβ and hTCR γδ. Cells are first gated for hCD3 and analyzed for TCR expression.

(F) Representative example of Human T cell amplification under hCD3 and hCD23 stimulation. The entire thymic population is labeled with CFSE at D0. During 5 days the labeled cells were cultured with or without hCD3 and hCD23. At D5, the unstimulated population is red, while the stimulated parent population is blue and the D0 population is green (n=6, 3 independent experiments).

(G) Confidence intervals of 1/(stem cell frequency) were calculated by ELDA (http://bioinf.wehi.edu.au/software/elda/) according to the Poisson distribution.

**Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.**

**Figure 5: In vivo engraftment of D17 EB cells in immunocompromised NSG mice, secondary recipients.**
(D) UMAP representation of single-cell RNA-Seq from NSG mouse L5 BM (14173 cells) transplanted with D17 EB cells for 20 weeks. Mouse and human cell clusters are identified.

(E-F) Bone marrow of NSG mouse L5 primary recipient analyzed by FACS at 20 weeks for (E) human erythroid cells (hCD235a⁺ and hCD235a+hCD71⁺) and (F) B cells (hIgM⁺CD10⁺), neutrophils (hCD33⁺hCD14⁺), and T cells (hCD4⁺hCD3⁺).

(G) Expression of globin genes in human early erythroid cells in transplanted mouse bone marrow datasets.

**Figure 4: Functional assessment of the grafted cells**

(A) Clonogenic hematopoietic assays on BM cells isolated from primary recipients grafted with either D17 EB (n=59, 14 independent experiments) or CD34⁺ CB cells (n=13, 3 independent experiments). Frequency of CFU-GM, BFU-E, and CFU-GEMM colonies.

(B) Representative colonies of CFU-GEMM (1), CFU-E (2) and CFU-GM (3) from D17 EB primary BM cells (n=59, 14 independent experiments).

(C) Cytospins. May Grünwald-Giemsa staining of cells isolated from clonogenic assays in primary or secondary recipients. Mature macrophages (1), histio monocytes (2), myelocytes (2), and erythroblasts (3). **B: bar=50µm; C: bar= 10µm**

(D) Human globin expression from CB CD34⁺HSPCs erythroid culture (n=3), from BM primary (n=12) and secondary recipients (n=4) and pooled BFU-E from BM primary recipients (6 to 10 BFU-E pooled. n=12, 3 independent experiments. Data are mean ± SEM

(E) Maturation of human T cells. Peripheral blood from a representative grafted primary recipient stained with antibodies against hTCR αβ and hTCR γδ. Cells are first gated for hCD3 and analyzed for TCR expression.

(F) Representative example of Human T cell amplification under hCD3 and hCD23 stimulation. The entire thymic population is labeled with CFSE at D0. During 5 days the labeled cells were cultured with or without hCD3 and hCD23. At D5, the unstimulated population is red, while the stimulated parent population is blue and the D0 population is green (n=6, 3 independent experiments).

(G) Confidence intervals of 1/(stem cell frequency) were calculated by ELDA (http://bioinf.wehi.edu.au/software/elda/) according to the Poisson distribution.

Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.

**Figure 5: In vivo engraftment of D17 EB cells in immunocompromised NSG mice, secondary recipients.**
(A) Flow cytometric analysis of human and mouse CD45⁺ cell expression in BM of a representative secondary recipient.

(B) *In vivo* engraftment of D17 human EB cells. All mixed hiPSC lines. Each circle/square/triangle represents a single animal. n= 40, 10 independent experiments.

(C) *In vivo* engraftment of D17 human EB cells from the five individual hiPSC lines: FD136-25 (n=10, 3 independent experiments, red circles), PC1429 (n=10, 3 independent experiments, orange squares), PCi-CAU (n=10, 3 independent experiments, green triangles), LAM001.005 (n=4, 2 independent experiments, light blue lozenges) and LAM002.002 (n=6, 2 independent experiments, dark blue lozenges) and the CD34⁺ CB cells (n=13, 3 independent experiments, grey circles) in NSG secondary recipients 20 weeks after transplantation of 7.10⁶ BM cells. FACS results are expressed as % of hCD34⁺, hCD43⁺, and hCD45⁺.

(D) Multilineage analysis. Human myeloid, B-lymphoid, and T-lymphoid cells (hCD33⁺, hCD19⁺, and hCD3⁺, respectively) and hCD235a⁺ erythroid progenitors/precursors 20 weeks after transplantation of the bone marrow from primary mice that received each of the five D17 EB cells compared with CD34⁺ CB cells. Same number of animals and presentation as in (C).

(E) Flow cytometric analysis of a representative secondary recipient transplanted with 7.10⁶ D17 EB cells. 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.

(F) Clonogenic hematopoietic assays on BM cells isolated from secondary recipients grafted with either D17 EB (n=10, 2 independent experiments) or CD34⁺ CB cells (n=13, 3 independent experiments). Frequency of CFU-GM, BFU-E, and CFU-GEMM colonies.

Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.

<caption>**Figure 6: Transcriptomic analysis of the EB cells**</caption>

(A) UMAP embeddings of cells in EBs at D13, 15, 17, and 19, 26027 cells. EVT: Extravillous
(A) Flow cytometric analysis of human and mouse CD45⁺ cell expression in BM of a representative secondary recipient.

(B) *In vivo* engraftment of D17 human EB cells. All mixed hiPSC lines. Each circle/square/triangle represents a single animal. **n= 40, 10 independent experiments.**

(C) *In vivo* engraftment of D17 human EB cells from the five individual hiPSC lines: FD136-25 (n=10, 3 independent experiments, red circles), PC1429 (n=10, 3 independent experiments, orange squares), PCi-CAU (n=10, 3 independent experiments, green triangles), LAM001.005 (n=4, 2 independent experiments, light blue lozenges) and LAM002.002 (n=6, 2 independent experiments, dark blue lozenges) and the CD34⁺ CB cells (n=13, 3 independent experiments, grey circles) in NSG secondary recipients 20 weeks after transplantation of 7.10⁶ BM cells. FACS results are expressed as % of hCD34⁺, hCD43⁺, and hCD45⁺.

(D) Multilineage analysis. Human myeloid, B-lymphoid, and T-lymphoid cells (hCD33⁺, hCD19⁺, and hCD3⁺, respectively) and hCD235a⁺ erythroid progenitors/precursors 20 weeks after transplantation of the bone marrow from primary mice that received each of the five D17 EB cells compared with CD34⁺ CB cells. Same number of animals and presentation as in (C).

(E) Flow cytometric analysis of a representative secondary recipient transplanted with 7.10⁶ D17 EB cells. 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.

(F) Clonogenic hematopoietic assays on BM cells isolated from secondary recipients grafted with either D17 EB (n=10, 2 independent experiments) or CD34⁺ CB cells (n=13, 3 independent experiments). Frequency of CFU-GM, BFU-E, and CFU-GEMM colonies.

Statistical data are presented as box plots and are mean ± SEM. Wilcoxon Mann-Whitney test.

<caption>**Figure 6: Transcriptomic analysis of the EB cells**</caption>

(A) UMAP embeddings of cells in EBs at D13, 15, 17, and 19, 26027 cells. EVT: Extravillous
Trophoblast. MEP: Megakaryocyte-Erythroid Progenitors. ECM: Extracellular Matrix. AGM: Aorta-Gonad-Mesonephros.

(B) Expression of genes in Crosse et al., 2020 characterizing endothelial populations in the AGM applied to the EB dataset

(C) UMAP embedding of cells in the data set of Crosse et al., 2020. HSPC: Hematopoietic stem and progenitor cells. PGC: Primordial germ cells. Hem: Hematopoietic cells. CL8: Cluster 8 described in Crosse et al., 2020⁵⁰.

(D) Expression of genes described in Crosse et al., 2020 ⁵⁰ characterizing endothelial populations in the AGM applied to Crosse et al. data set.

(E) Close-up of the UMAP embedding on the hematopoietic cell populations in the EB dataset framed in A. MEP: Megakaryocyte-Erythroid Progenitors. HSC: Hematopoietic Stem Cells. HE: Hemogenic Endothelium. Erythro: Erythroid cells.

(F) Expression of the six-gene molecular HSC signature of Calvanese et al., 2022 ⁵² in the hematopoietic population of the embryoid body dataset.

**Figure 7: Molecular characterization of the cells in the embryoid bodies**

(A-C) Time-course detection of molecular markers of endothelial and hematopoietic cell populations between scRNA-Seq and q-PCR datasets comparison (A), in FACS (B) and scRNA-Seq (C).

(D-G) Immunostaining on transverse sections of D17 EBs showing spatial organization of endothelium and peri-endothelial populations. (D) Endothelial cells (CDH5). (E, F) cells expressing PECAM1 and the transcription factor RUNX1. (G) Pericytes (NG2⁺) and smooth muscle cells (αSMA⁺).

D, E, F, G: Bars=100 microns.
Trophoblast. MEP: Megakaryocyte-Erythroid Progenitors. ECM: Extracellular Matrix. AGM: Aorta-Gonad-Mesonephros.

(B) Expression of genes in Crosse et al., 2020 characterizing endothelial populations in the AGM applied to the EB dataset

(C) UMAP embedding of cells in the data set of Crosse et al., 2020. HSPC: Hematopoietic stem and progenitor cells. PGC: Primordial germ cells. Hem: Hematopoietic cells. CL8: Cluster 8 described in Crosse et al., 2020⁵⁰.

(D) Expression of genes described in Crosse et al., 2020 ⁵⁰ characterizing endothelial populations in the AGM applied to Crosse et al. data set.

(E) Close-up of the UMAP embedding on the hematopoietic cell populations in the EB dataset framed in A. MEP: Megakaryocyte-Erythroid Progenitors. HSC: Hematopoietic Stem Cells. HE: Hemogenic Endothelium. Erythro: Erythroid cells.

(F) Expression of the six-gene molecular HSC signature of Calvanese et al., 2022 ⁵² in the hematopoietic population of the embryoid body dataset.

**Figure 7: Molecular characterization of the cells in the embryoid bodies**

(A-C) Time-course detection of molecular markers of endothelial and hematopoietic cell populations between scRNA-Seq and q-PCR datasets comparison (A), in FACS (B) and scRNA-Seq (C).

(D-G) Immunostaining on transverse sections of D17 EBs showing spatial organization of endothelium and peri-endothelial populations. (D) Endothelial cells (CDH5). (E, F) cells expressing PECAM1 and the transcription factor RUNX1. (G) Pericytes (NG2⁺) and smooth muscle cells (αSMA⁺).

D, E, F, G: Bars=100 microns.
* Please include these specific headings in the following order: RESOURCE AVAILABILITY; EXPERIMENTAL MODEL AND SUBJECT DETAILS; METHOD DETAILS; QUANTIFICATION AND STATISTICAL ANALYSIS and add all the relevant information, as further explained in the attached document.

# STAR★Methods

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information should be directed to and will be fulfilled by the lead contact, Laurence Guyonneau-Harmand (laurence.harmand@upmc.fr).

### Materials availability

This study did not generate any unique reagents.

### Data and code availability

* Single-cell RNA-seq data have been deposited at the National Center for Biotechnology Information BioProjects Gene Expression Omnibus (GEO) under accession number GEO:GSE151877. Accession numbers are listed in the key resource table.

* *In vivo* single-cell RNAseq data from the human embryo were from Calvanese et al.,<sup>53</sup> and Zeng et al.<sup>54</sup>

* No original code is reported in this paper.

* Any additional information required for re-analysis of the data reported in this paper is available on request from the lead contact.
* Please include these specific headings in the following order: RESOURCE AVAILABILITY; EXPERIMENTAL MODEL AND SUBJECT DETAILS; METHOD DETAILS; QUANTIFICATION AND STATISTICAL ANALYSIS and add all the relevant information, as further explained in the attached document.

# STAR★Methods

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information should be directed to and will be fulfilled by the lead contact, Laurence Guyonneau-Harmand (laurence.harmand@upmc.fr).

### Materials availability

This study did not generate any unique reagents.

### Data and code availability

* Single-cell RNA-seq data have been deposited at the National Center for Biotechnology Information BioProjects Gene Expression Omnibus (GEO) under accession number GEO:GSE151877. Accession numbers are listed in the key resource table.

* *In vivo* single-cell RNAseq data from the human embryo were from Calvanese et al.,<sup>53</sup> and Zeng et al.<sup>54</sup>

* No original code is reported in this paper.

* Any additional information required for re-analysis of the data reported in this paper is available on request from the lead contact.
# EXPERIMENTAL MODEL AND PARTICIPANT DETAILS

## Ethics statement

All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation and approved by the local ethics committee (APAFIS approval number # 17559-2018111613396032v2 (2024)).

## Animals

Two animal models were used for this study:

- NOD/SCID-LtSz-scid/scid (NOD/SCID) (Charles River, L'Abresle, France)

- NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ or NSG (Charles River, L'Abresle, France)

We choose to work with female mice because they are less aggressive and this is an important factor when the experience lasts 20 weeks with regular sampling.

## Cell lines

For cell lines, primary cultures, and microbe strains, please describe culture/growth conditions, including temperature. Sex of cells must also be reported

MS-5 [Mouse bone marrow] (RRID:CVCL_2128): mouse male cells are maintained in 90% alpha-MEM with ribo- and deoxyribonucleosides + 10% decomplemented FBS + 2 mM L-glutamine + 2 mM sodium pyruvate at 37°C and 5% CO₂.

PCi-CAU : human male induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

PCi-1429 : human female induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

IMR90-16 : human female induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.
# EXPERIMENTAL MODEL AND PARTICIPANT DETAILS

## Ethics statement

All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation and approved by the local ethics committee (APAFIS approval number # 17559-2018111613396032v2 (2024)).

## Animals

Two animal models were used for this study:

- NOD/SCID-LtSz-scid/scid (NOD/SCID) (Charles River, L'Abresle, France)

- NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ or NSG (Charles River, L'Abresle, France)

We choose to work with female mice because they are less aggressive and this is an important factor when the experience lasts 20 weeks with regular sampling.

## Cell lines

For cell lines, primary cultures, and microbe strains, please describe culture/growth conditions, including temperature. Sex of cells must also be reported

MS-5 [Mouse bone marrow] (RRID:CVCL_2128): mouse male cells are maintained in 90% alpha-MEM with ribo- and deoxyribonucleosides + 10% decomplemented FBS + 2 mM L-glutamine + 2 mM sodium pyruvate at 37°C and 5% CO₂.

PCi-CAU : human male induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

PCi-1429 : human female induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

IMR90-16 : human female induced pluripotent stem cells are maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.
FD136-25 : human female induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

LAM001.005 : human female induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

LAM002.002 human male induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

Cord blood CD34⁺: We obtain cord blood units from AP-HP, Hôpital Saint-Louis, Unité de Thérapie cellulaire, CRB-Banque de Sang de Cordon, Paris, France(n° d'autorisation: AC-2016-2759) for research purposes only. They are therefore anonymized and the sex of the baby is not mentioned. CD34⁺ are sorted and transplanted or seeded in methylcellulose with BSA, IMDM and SCF, IL-3, EPO and GM-CSF.

## METHOD DETAILS

### hiPSC amplification

The study was conducted using six different hiPSC lines: the FD136-25 (skin primary fibroblasts) reprogrammed with lentiviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28), IMR90-16 (fetal lung fibroblasts) reprogrammed with lentiviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28) ², the Pci-1429 and Pci-CAU lines (peripheral blood mononuclear cells-Phenocell) reprogrammed with episomes (Sox2, Oct4, KLF, cMyc); the LAM001-005 and LAM002-002 lines (skin primary fibroblasts), reprogrammed with sendaï virus (Oct3/4, Nanog, KLF and cMyc) by IPS CDTC core facility, Nantes, France. hiPSCs were maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) and the cells were passaged 1:6 onto freshly coated plates every 3 to 5 days using standard clump passaging with TRYple select (Invitrogen) or ReLeSR (Stemcell Technologies).

ABG ATMP platform worked with Pci-CAU line and used vitronectin matrix (Gibco™), IPSbrew medium (Miltenyi Biotec) and ReLeSR (Stemcell Technologies) for passaging.

## Design of experiment
FD136-25 : human female induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

LAM001.005 : human female induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

LAM002.002 human male induced pluripotent stem cells are maintained on Matrigel™
(Corning) in mTESRplus medium (Stemcell Technologies) at 37°C and 5% CO₂.

Cord blood CD34⁺: We obtain cord blood units from AP-HP, Hôpital Saint-Louis, Unité de Thérapie cellulaire, CRB-Banque de Sang de Cordon, Paris, France(n° d'autorisation: AC-2016-2759) for research purposes only. They are therefore anonymized and the sex of the baby is not mentioned. CD34⁺ are sorted and transplanted or seeded in methylcellulose with BSA, IMDM and SCF, IL-3, EPO and GM-CSF.

## METHOD DETAILS

### hiPSC amplification

The study was conducted using six different hiPSC lines: the FD136-25 (skin primary fibroblasts) reprogrammed with lentiviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28), IMR90-16 (fetal lung fibroblasts) reprogrammed with lentiviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28) ², the Pci-1429 and Pci-CAU lines (peripheral blood mononuclear cells-Phenocell) reprogrammed with episomes (Sox2, Oct4, KLF, cMyc); the LAM001-005 and LAM002-002 lines (skin primary fibroblasts), reprogrammed with sendaï virus (Oct3/4, Nanog, KLF and cMyc) by IPS CDTC core facility, Nantes, France. hiPSCs were maintained on Matrigel™ (Corning) in mTESRplus medium (Stemcell Technologies) and the cells were passaged 1:6 onto freshly coated plates every 3 to 5 days using standard clump passaging with TRYple select (Invitrogen) or ReLeSR (Stemcell Technologies).

ABG ATMP platform worked with Pci-CAU line and used vitronectin matrix (Gibco™), IPSbrew medium (Miltenyi Biotec) and ReLeSR (Stemcell Technologies) for passaging.

## Design of experiment
EBs were cultured in liquid medium IMDM supplemented with stabilized glutamine (SAFC), 330 µg/mL holo-human transferrin (Scipac), 10 µg/mL recombinant human insulin (Incelligent SG; CellGen), 2 IU/mL heparin Choay, and 5% solvent/detergent virus-inactivated AB plasma (Etablissement Français du Sang). This plasma is used in a clinical trial registered at http://www.clinicaltrials.gov as NCT0929266. Plasma samples can be obtained upon reasonable request.

Optimal response surface modeling was applied to determine the optimal concentrations of the 10 selected cytokines. A two-factorial experimental design was chosen with a range of 10 independent variables. The following cytokines were selected on the basis of personal experience and data from the literature: SCF (20 to 300 ng/mL), IGF-1 (5 to 50 ng/mL), TPO (20 to 100 ng/mL), IL1 (5 to 50 ng/mL), IL3 (5 to 50 ng/mL), IL6 (5 to 50 ng/mL), G-CSF (10 to 100 ng/mL), BMP4 (20 to 200 ng/mL), VEGF (20 to 200 ng/mL) and FLT3 (20 to 300 ng/mL) (Table S1). Human iPSCs were cultured for 17 days using 16 different combinations of cytokines. In each case, the cells were phenotyped and their amplification rate and their ability to generate LTC-ICs were determined (table S1). Statistical design, regression analysis of the results and plotting of the response surface were performed using the software JMP-8 (SAS Institute Inc., Cary, USA). The software generated an algorithm which identified three efficient combinations of cytokines (table S2).

## EB differentiation

The EB culture medium (see above) was supplemented with stem cell factor (24 ng/mL), thrombopoietin (21 ng/mL), FLT3 ligand (21 ng/mL), recombinant human bone morphogenetic protein 4 (194 ng/mL), recombinant human vascular endothelial growth factor (200 ng/mL), interleukin-3 (50 ng/mL), interleukin-6 (50 ng/mL), interleukin-1 (5 ng/mL), granulocyte colony-stimulating factor (100 ng/mL) and insulin growth factor (1 ng/mL) (GMP grade when available otherwise Premium grade, Peprotech). The medium was replenished every other day until 19 days at 37°C in a humidified 5% CO₂ atmosphere. EBs were dissociated in 0.1% collagenase B (Roche Diagnostics, Laval, QC, Canada) for 1 hour at 37°C then 0.25% trypsin for 3 min and disrupted by gentle mechanical agitation

## Colony assays
EBs were cultured in liquid medium IMDM supplemented with stabilized glutamine (SAFC), 330 µg/mL holo-human transferrin (Scipac), 10 µg/mL recombinant human insulin (Incelligent SG; CellGen), 2 IU/mL heparin Choay, and 5% solvent/detergent virus-inactivated AB plasma (Etablissement Français du Sang). This plasma is used in a clinical trial registered at http://www.clinicaltrials.gov as NCT0929266. Plasma samples can be obtained upon reasonable request.

Optimal response surface modeling was applied to determine the optimal concentrations of the 10 selected cytokines. A two-factorial experimental design was chosen with a range of 10 independent variables. The following cytokines were selected on the basis of personal experience and data from the literature: SCF (20 to 300 ng/mL), IGF-1 (5 to 50 ng/mL), TPO (20 to 100 ng/mL), IL1 (5 to 50 ng/mL), IL3 (5 to 50 ng/mL), IL6 (5 to 50 ng/mL), G-CSF (10 to 100 ng/mL), BMP4 (20 to 200 ng/mL), VEGF (20 to 200 ng/mL) and FLT3 (20 to 300 ng/mL) (Table S1). Human iPSCs were cultured for 17 days using 16 different combinations of cytokines. In each case, the cells were phenotyped and their amplification rate and their ability to generate LTC-ICs were determined (table S1). Statistical design, regression analysis of the results and plotting of the response surface were performed using the software JMP-8 (SAS Institute Inc., Cary, USA). The software generated an algorithm which identified three efficient combinations of cytokines (table S2).

## EB differentiation

The EB culture medium (see above) was supplemented with stem cell factor (24 ng/mL), thrombopoietin (21 ng/mL), FLT3 ligand (21 ng/mL), recombinant human bone morphogenetic protein 4 (194 ng/mL), recombinant human vascular endothelial growth factor (200 ng/mL), interleukin-3 (50 ng/mL), interleukin-6 (50 ng/mL), interleukin-1 (5 ng/mL), granulocyte colony-stimulating factor (100 ng/mL) and insulin growth factor (1 ng/mL) (GMP grade when available otherwise Premium grade, Peprotech). The medium was replenished every other day until 19 days at 37°C in a humidified 5% CO₂ atmosphere. EBs were dissociated in 0.1% collagenase B (Roche Diagnostics, Laval, QC, Canada) for 1 hour at 37°C then 0.25% trypsin for 3 min and disrupted by gentle mechanical agitation

## Colony assays
At the indicated times, 10⁵ dissociated EB cells or 3.10⁴ cells from xenotransplanted recipient BM were plated into 3 mL of complete methylcellulose medium in the presence of SCF, IL-3, EPO and GM-CSF (PeproTech, Neuilly-sur-Seine, France). As G-CSF also stimulates mouse progenitors, it was replaced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Aliquots (1 mL) of the mix were distributed into one 30 mm dish twice and maintained in a humidified chamber for 14 days. Colony-forming Cells (CFC) were scored on day 14.

## Long-term culture-initiating cell assay

Long-term culture-initiating cell (LTC-IC) assays were performed as described previously ⁶⁰, 15–100,000 cells/well on day 17 for the EBs and on day 0 for the control CD34⁺. Absolute LTC-IC counts corresponded to the cell concentrations, yielding 37% negative wells using Poisson statistics.

## Pseudo-microtubules

Growth factor-reduced Matrigel (Becton Dickinson, New Jersey, USA) was thawed, placed in 24-well plates, and allowed to stand at 37°C for 30 minutes to solidify. Aliquots of 5x10⁴ hiPSC cells/well were plated in 1 ml EBF2 medium (Lonza, Walkersville, USA) and incubated at 37°C for one week. A tubule is defined as a structure whose length is four times its width. The presence of tubules was checked daily, and they usually form within the first 6 hours.

## Flow cytometry

Staining of hiPSCs or dissociated EBs or mouse tissue cells was performed with 2-5.10⁵ cells in 100 µL staining buffer (PBS containing 2% FBS and FcR Blocking Reagent) with 5:100 dilution of each antibody, for 20 min at room temperature in the dark. Viability staining was performed using either DAPI or the Zombie aqua fixable viability kit (Biolegend). Data were collected using a Becton Dickinson Canto II cytometer or a MACS Quant VyB or a MACS Quant 10 (Miltenyi).

Three labs analyzed the data, the list of antibodies is given in "Key resources methods".

## Real-Time Quantitative PCR

After isolation of total mRNA with an RNA minikit (Qiagen, Courtaboeuf, France), the mRNA integrity was checked by analysis on a Bioanalyzer 2100 (Agilent Technologies, Massy, France).
At the indicated times, 10⁵ dissociated EB cells or 3.10⁴ cells from xenotransplanted recipient BM were plated into 3 mL of complete methylcellulose medium in the presence of SCF, IL-3, EPO and GM-CSF (PeproTech, Neuilly-sur-Seine, France). As G-CSF also stimulates mouse progenitors, it was replaced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Aliquots (1 mL) of the mix were distributed into one 30 mm dish twice and maintained in a humidified chamber for 14 days. Colony-forming Cells (CFC) were scored on day 14.

## Long-term culture-initiating cell assay

Long-term culture-initiating cell (LTC-IC) assays were performed as described previously ⁶⁰, 15–100,000 cells/well on day 17 for the EBs and on day 0 for the control CD34⁺. Absolute LTC-IC counts corresponded to the cell concentrations, yielding 37% negative wells using Poisson statistics.

## Pseudo-microtubules

Growth factor-reduced Matrigel (Becton Dickinson, New Jersey, USA) was thawed, placed in 24-well plates, and allowed to stand at 37°C for 30 minutes to solidify. Aliquots of 5x10⁴ hiPSC cells/well were plated in 1 ml EBF2 medium (Lonza, Walkersville, USA) and incubated at 37°C for one week. A tubule is defined as a structure whose length is four times its width. The presence of tubules was checked daily, and they usually form within the first 6 hours.

## Flow cytometry

Staining of hiPSCs or dissociated EBs or mouse tissue cells was performed with 2-5.10⁵ cells in 100 µL staining buffer (PBS containing 2% FBS and FcR Blocking Reagent) with 5:100 dilution of each antibody, for 20 min at room temperature in the dark. Viability staining was performed using either DAPI or the Zombie aqua fixable viability kit (Biolegend). Data were collected using a Becton Dickinson Canto II cytometer or a MACS Quant VyB or a MACS Quant 10 (Miltenyi).

Three labs analyzed the data, the list of antibodies is given in "Key resources methods".

## Real-Time Quantitative PCR

After isolation of total mRNA with an RNA minikit (Qiagen, Courtaboeuf, France), the mRNA integrity was checked by analysis on a Bioanalyzer 2100 (Agilent Technologies, Massy, France).
Complementary DNAs were constructed by reverse transcription with Superscript (Life Technologies, Carlsbad, USA). PCR assays were performed using a TaqMan PCR master mix (Life Technologies) and specific primers (Applied BioSystems, Carlsbad, USA) for selected genes (see table below), together with a sequence detection system (QuantStudio™ 12K Flex Real-Time PCR System, Life Technologies). In each sample the fluorescent PCR signal of each target gene was normalized to the fluorescent signal of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

## Mouse transplantation

NOD/SCID-LtSz-scid/scid (NOD/SCID) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) (Charles River, L'Abresle, France) were housed in the IRSN animal care facility. All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation and approved by

the local ethics committee (APAFIS approval number # 17559-2018111613396032v2 (2024)).

Mice, 5-8 weeks old and raised under sterile conditions, were sublethally irradiated with 2.4-3.5 Grays from a 137Cs source (2.115 Gy/min) 24 h before cell injection. To ensure consistency between experiments, only male mice were used. Prior to transplantation, the mice were temporarily sedated with an intraperitoneal injection of ketamine and xylazine. In primary recipients, 4 x10⁵ D17 or CD34⁺ CB cells (cord blood obtained from AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, CRB-Banque de Sang de Cordon, Paris, France (authorization number : AC-2016-2759) per mouse were transplanted by retro-orbital injection in a volume of 100 µl with a 28.5-gauge insulin needle. Secondary recipients were transplanted with 7,10⁶ BM cells under the same conditions as the primary mice. A total of 251 mice were used in this study.

For the engraftment potential of the D17 cells on the five different hiPSC lines:

205 NSG mice were used as follows: 113 primary recipients, 53 secondary recipients and 39 as negative controls.
Complementary DNAs were constructed by reverse transcription with Superscript (Life Technologies, Carlsbad, USA). PCR assays were performed using a TaqMan PCR master mix (Life Technologies) and specific primers (Applied BioSystems, Carlsbad, USA) for selected genes (see table below), together with a sequence detection system (QuantStudio™ 12K Flex Real-Time PCR System, Life Technologies). In each sample the fluorescent PCR signal of each target gene was normalized to the fluorescent signal of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

## Mouse transplantation

NOD/SCID-LtSz-scid/scid (NOD/SCID) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) (Charles River, L'Abresle, France) were housed in the IRSN animal care facility. All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation and approved by

the local ethics committee (APAFIS approval number # 17559-2018111613396032v2 (2024)).

Mice, 5-8 weeks old and raised under sterile conditions, were sublethally irradiated with 2.4-3.5 Grays from a 137Cs source (2.115 Gy/min) 24 h before cell injection. To ensure consistency between experiments, only male mice were used. Prior to transplantation, the mice were temporarily sedated with an intraperitoneal injection of ketamine and xylazine. In primary recipients, 4 x10⁵ D17 or CD34⁺ CB cells (cord blood obtained from AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, CRB-Banque de Sang de Cordon, Paris, France (authorization number : AC-2016-2759) per mouse were transplanted by retro-orbital injection in a volume of 100 µl with a 28.5-gauge insulin needle. Secondary recipients were transplanted with 7,10⁶ BM cells under the same conditions as the primary mice. A total of 251 mice were used in this study.

For the engraftment potential of the D17 cells on the five different hiPSC lines:

205 NSG mice were used as follows: 113 primary recipients, 53 secondary recipients and 39 as negative controls.
63 NOD-SCID mice were used as follows: 35 primary recipients and 16 secondary recipients, 3 tertiary recipients and 9 as negative controls (see Table S3). 4.10⁵, 7.10⁶ and 7.10⁶ cells were inoculated for primary, secondary and tertiary recipients respectively.

Animals were analyzed 20 weeks after primary, secondary and even tertiary transplantation, with the exception of those processed on the ATM platform. These were analyzed after 8 and 12 weeks.

## Limiting dilution.

For the limiting dilution assay (LDA), 33 NSG mice were used, i.e., 30 primary recipients and 3 controls. The number of human HSCs was determined by LDA. Increasing doses of d17 cells (1000, 5000, 10000, 50000, 100000) were retro-orbitally injected into sublethally irradiated NSG recipient mice. Twenty weeks after transplantation, the percentage of hCD45⁺ cells were analyzed by flow cytometry. HSC frequency was calculated and recorded using ELDA software⁶¹ (bioinf.wehi.edu.au/software/elda/).

## Assessment of human cell engraftment

Most of the mice were sacrificed at week 20. A few of them were sacrificed at 8 or 12 weeks. Femurs, tibias, blood, liver, spleen and thymus were removed. Single cell suspensions were prepared by standard flushing and aliquots containing 7.10⁵ cells were stained in a total volume of 100µL staining buffer (see also "Flow cytometry"). Non-injected mouse BM was used as a control for non-specific staining.

Data were acquired on a BD Canto II cytometer (Beckton Dinkinson), MACSQuant 10 and MACSQuant VYB (Miltenyi).

## T-cell maturity and functionality assay

The presence of TCR αβ and TCR γδ in peripheral blood was assessed by flow cytometry using the following human markers: hCD3 clone UCHT1 (positive gating), TCR αβ clone IP26A and TCR γδ clone IMMU510 (all from Beckman Coulter antibodies, Brea, USA).

Thymus and spleen cells were isolated, CFSE labeled (cell trace CFSE cell proliferation kit, Thermofisher Scientific) and seeded in cell culture media complemented or not with hCD3 and
63 NOD-SCID mice were used as follows: 35 primary recipients and 16 secondary recipients, 3 tertiary recipients and 9 as negative controls (see Table S3). 4.10⁵, 7.10⁶ and 7.10⁶ cells were inoculated for primary, secondary and tertiary recipients respectively.

Animals were analyzed 20 weeks after primary, secondary and even tertiary transplantation, with the exception of those processed on the ATM platform. These were analyzed after 8 and 12 weeks.

## Limiting dilution.

For the limiting dilution assay (LDA), 33 NSG mice were used, i.e., 30 primary recipients and 3 controls. The number of human HSCs was determined by LDA. Increasing doses of d17 cells (1000, 5000, 10000, 50000, 100000) were retro-orbitally injected into sublethally irradiated NSG recipient mice. Twenty weeks after transplantation, the percentage of hCD45⁺ cells were analyzed by flow cytometry. HSC frequency was calculated and recorded using ELDA software⁶¹ (bioinf.wehi.edu.au/software/elda/).

## Assessment of human cell engraftment

Most of the mice were sacrificed at week 20. A few of them were sacrificed at 8 or 12 weeks. Femurs, tibias, blood, liver, spleen and thymus were removed. Single cell suspensions were prepared by standard flushing and aliquots containing 7.10⁵ cells were stained in a total volume of 100µL staining buffer (see also "Flow cytometry"). Non-injected mouse BM was used as a control for non-specific staining.

Data were acquired on a BD Canto II cytometer (Beckton Dinkinson), MACSQuant 10 and MACSQuant VYB (Miltenyi).

## T-cell maturity and functionality assay

The presence of TCR αβ and TCR γδ in peripheral blood was assessed by flow cytometry using the following human markers: hCD3 clone UCHT1 (positive gating), TCR αβ clone IP26A and TCR γδ clone IMMU510 (all from Beckman Coulter antibodies, Brea, USA).

Thymus and spleen cells were isolated, CFSE labeled (cell trace CFSE cell proliferation kit, Thermofisher Scientific) and seeded in cell culture media complemented or not with hCD3 and
hCD28 (Beckman Coulter both 1mg/ml). After 5 days, cells were harvested and stained with anti-hCD3 clone UCHT1 and analyzed on a BD Canto II cytometer. FlowJo analysis software was used to gate on CD3+T-cells and generate the overlaid histogram plots.

## Cell preparation for sequencing

PCi-CAU EB cell suspensions at different times were loaded on a Chromium single cell instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs and prepared libraries according to the manufacturer's recommendations and performed Illumina Sequencing. Experiments were done in duplicates.

## ScRNA-Seq analysis of mouse bone marrow

Both samples were aligned to mm10 and grch38 using CellRanger 6.1.2. For each sample, we selected only the cells expressing at least 200 genes in both genome assemblies. We identified the mouse cells from the human cells as described in the section "Separation strategy for human and mouse cells in scRNA-Seq" and filtered out the duplicates. Cells expressing less than 5000 genes on grch38 and 6000 genes on mm10 were retained. Cells expressing more than 10% of human mitochondrial RNA or 4% of mouse mitochondrial RNA were filtered out. The merged dataset from the two mice was then normalized using SCTransform with default parameters. UMAP visualization (n neighbors=100) and Louvain clustering at 0.6 resolution were then performed for the first 38 PCA components. The same process was used for the mouse L5 and human cell plots, except for the number of PCA components selected, which varied: 39 for mouse L5 and 40 for human cells. Cell type identification was performed for the merged dataset of the two bone marrow cells with differentially expressed genes using SoupX tf-idf gene lists, scHCL and scMCA mapping, and manual examination of marker genes.

## Separation strategy for human and mouse cells in BM scRNA-Seq

As a control, the human (SRR7881423) and mouse (SRR6835854) public BM datasets were aligned to mm10 and grch38 using CellRanger 6.0.0. Only cells that met CellRanger's filter threshold and had at least 200 features in grch38 or mm10 were selected for further analysis. All cells were then pooled and analyzed using the standard Seurat SCTransform pipeline for
hCD28 (Beckman Coulter both 1mg/ml). After 5 days, cells were harvested and stained with anti-hCD3 clone UCHT1 and analyzed on a BD Canto II cytometer. FlowJo analysis software was used to gate on CD3+T-cells and generate the overlaid histogram plots.

## Cell preparation for sequencing

PCi-CAU EB cell suspensions at different times were loaded on a Chromium single cell instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs and prepared libraries according to the manufacturer's recommendations and performed Illumina Sequencing. Experiments were done in duplicates.

## ScRNA-Seq analysis of mouse bone marrow

Both samples were aligned to mm10 and grch38 using CellRanger 6.1.2. For each sample, we selected only the cells expressing at least 200 genes in both genome assemblies. We identified the mouse cells from the human cells as described in the section "Separation strategy for human and mouse cells in scRNA-Seq" and filtered out the duplicates. Cells expressing less than 5000 genes on grch38 and 6000 genes on mm10 were retained. Cells expressing more than 10% of human mitochondrial RNA or 4% of mouse mitochondrial RNA were filtered out. The merged dataset from the two mice was then normalized using SCTransform with default parameters. UMAP visualization (n neighbors=100) and Louvain clustering at 0.6 resolution were then performed for the first 38 PCA components. The same process was used for the mouse L5 and human cell plots, except for the number of PCA components selected, which varied: 39 for mouse L5 and 40 for human cells. Cell type identification was performed for the merged dataset of the two bone marrow cells with differentially expressed genes using SoupX tf-idf gene lists, scHCL and scMCA mapping, and manual examination of marker genes.

## Separation strategy for human and mouse cells in BM scRNA-Seq

As a control, the human (SRR7881423) and mouse (SRR6835854) public BM datasets were aligned to mm10 and grch38 using CellRanger 6.0.0. Only cells that met CellRanger's filter threshold and had at least 200 features in grch38 or mm10 were selected for further analysis. All cells were then pooled and analyzed using the standard Seurat SCTransform pipeline for
grch38 data: SCTransform (default parameters), RunPCA, RunUMAP (first 40 PCA dimensions, n.neighbors=100).

As shown in Figure S2E, F, the ratio between the number of features detected in grch38 and mm10 assemblies was sufficient to clearly distinguish all human cells from mouse cells. In addition, the human and mouse datasets are separated in the final UMAP plot (Figure S2I).

In the mouse transplanted bone marrow dataset, although two populations could be separated based on the feature ratio of grch38/mm10, some cells had an intermediate ratio (Figure S2G, H). Because no cell type in the control exhibited such a phenotype and these intermediate cells were scattered across the different UMAP populations (Figure S2J), we considered them duplicates and filtered them out, resulting in the plot shown in Figure S2K.

The human cells were those that had a ratio greater than 2.5 for mouse L1 and 2.85 for mouse L5. The mouse cells were those that had a ratio of less than 0.55 for mouse L1 and 0.47 for mouse L5. The total number of cells filtered (8%) was consistent with the expected number of duplicates using 10X Chromium technology.

## ScRNA-Seq analysis of the embryoid bodies

Each individual sample was filtered to obtain only genes expressed in at least 3 cells and cells that expressed between 200 and 8000 genes. Due to a higher percentage of dead cells, only cells with more than 1300 genes were selected for one of the D15 datasets (J15-TJ3). Cells expressing more than 10% mitochondrial RNA were filtered out. The merged dataset was then normalized using SCTransform with default parameters. UMAP visualization and Leiden clustering with a resolution of 1.2 were then performed for the first 82 PCA components, the latter generated with the parameter approx= FALSE in Seurat. To identify cell type, differentially expressed genes were used with the Wilcoxon assay of Seurat, the tf-idf of SoupX, and the scHCL in addition to manual examination of marker genes to confirm cell identity.

To more accurately identify the cell type of hematopoietic cells, cells with a UMAP_1 coordinate less than -7 and a UMAP_2 coordinate greater than -1 were extracted and renormalized using SCTransform with default parameters. Leiden clustering was then performed at a resolution of 1.3 on the first 35 PCA dimensions. Cell type identification followed the same procedure as described for the entire dataset.
grch38 data: SCTransform (default parameters), RunPCA, RunUMAP (first 40 PCA dimensions, n-neighbors=100).

As shown in Figure S2E, F, the ratio between the number of features detected in grch38 and mm10 assemblies was sufficient to clearly distinguish all human cells from mouse cells. In addition, the human and mouse datasets are separated in the final UMAP plot (Figure S2I).

In the mouse transplanted bone marrow dataset, although two populations could be separated based on the feature ratio of grch38/mm10, some cells had an intermediate ratio (Figure S2G, H). Because no cell type in the control exhibited such a phenotype and these intermediate cells were scattered across the different UMAP populations (Figure S2J), we considered them duplicates and filtered them out, resulting in the plot shown in Figure S2K.

The human cells were those that had a ratio greater than 2.5 for mouse L1 and 2.85 for mouse L5. The mouse cells were those that had a ratio of less than 0.55 for mouse L1 and 0.47 for mouse L5. The total number of cells filtered (8%) was consistent with the expected number of duplicates using 10X Chromium technology.

## ScRNA-Seq analysis of the embryoid bodies

Each individual sample was filtered to obtain only genes expressed in at least 3 cells and cells that expressed between 200 and 8000 genes. Due to a higher percentage of dead cells, only cells with more than 1300 genes were selected for one of the D15 datasets (J15-TJ3). Cells expressing more than 10% mitochondrial RNA were filtered out. The merged dataset was then normalized using SCTransform with default parameters. UMAP visualization and Leiden clustering with a resolution of 1.2 were then performed for the first 82 PCA components, the latter generated with the parameter approx= FALSE in Seurat. To identify cell type, differentially expressed genes were used with the Wilcoxon assay of Seurat, the tf-idf of SoupX, and the scHCL in addition to manual examination of marker genes to confirm cell identity.

To more accurately identify the cell type of hematopoietic cells, cells with a UMAP_1 coordinate less than -7 and a UMAP_2 coordinate greater than -1 were extracted and renormalized using SCTransform with default parameters. Leiden clustering was then performed at a resolution of 1.3 on the first 35 PCA dimensions. Cell type identification followed the same procedure as described for the entire dataset.
## Immunostaining of the embryoid bodies

For immunofluorescence analysis (IF), EBs were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature and sectioned using a Leica cryostat CM3050. Samples were then incubated for 60 minutes at room temperature with IF buffer [phosphate-buffered saline (PBS), 0.2% Triton, 5% FBS], which also served as a blocking solution. Samples were incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: anti-alpha SMA (1:500; Sigma-Aldrich A2547), anti-NG2 (1:400, Cell Signaling E3B3G), anti-VECAD (1:400, Cell Signaling D87F2), anti-CD31 (1:500, Cell Signaling 89C2) and anti-RUNX1 (1:100, Abcamab 92336). Incubation with the secondary antibodies (1:500, Invitrogen A-21121, Jackson 711-605-152) was performed for 1 hour at room temperature. Finally, 40,6-diamidino-2-phenylindole (DAPI) nuclear staining was performed (1:500 for 5 minutes). Immunofluorescence images were acquired using a Leica DMi 6000B microscope. Images were processed using ImageJ software.

## QUANTIFICATION AND STATISTICAL ANALYSIS

JMP-8 (SAS Institute Inc., Cary, USA) was used for DOE; the procedure is fully described in the Star Method above.

Confidence intervals of “LTC-IC frequency” and confidence intervals of “cell capable of graft frequency” were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution.

Graft quantification procedures are described in the Star Method. Criteria for successful engraftment were defined as >0.1% hCD45⁺ cells in mouse BM. In the figure legend, we define “n” as the number of individual mice and indicate the number of biological replicates.

We did not exclude any animals.

In the text, we have chosen to present means +/- SEM, while in the figures we have chosen to present box plots, which are useful for visualizing the distribution of the data across its quartiles.

The list and versions of softwares, patches used for scRNA-Seq analysis will be provided upon request.

Statistical analyses were performed using GraphPad Prism 8 and Excel.
## Immunostaining of the embryoid bodies

For immunofluorescence analysis (IF), EBs were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature and sectioned using a Leica cryostat CM3050. Samples were then incubated for 60 minutes at room temperature with IF buffer [phosphate-buffered saline (PBS), 0.2% Triton, 5% FBS], which also served as a blocking solution. Samples were incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: anti-alpha SMA (1:500; Sigma-Aldrich A2547), anti-NG2 (1:400, Cell Signaling E3B3G), anti-VECAD (1:400, Cell Signaling D87F2), anti-CD31 (1:500, Cell Signaling 89C2) and anti-RUNX1 (1:100, Abcamab 92336). Incubation with the secondary antibodies (1:500, Invitrogen A-21121, Jackson 711-605-152) was performed for 1 hour at room temperature. Finally, 40,6-diamidino-2-phenylindole (DAPI) nuclear staining was performed (1:500 for 5 minutes). Immunofluorescence images were acquired using a Leica DMi 6000B microscope. Images were processed using ImageJ software.

## QUANTIFICATION AND STATISTICAL ANALYSIS

JMP-8 (SAS Institute Inc., Cary, USA) was used for DOE; the procedure is fully described in the Star Method above.

Confidence intervals of “LTC-IC frequency” and confidence intervals of “cell capable of graft frequency” were calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution.

Graft quantification procedures are described in the Star Method. Criteria for successful engraftment were defined as >0.1% hCD45⁺ cells in mouse BM. In the figure legend, we define “n” as the number of individual mice and indicate the number of biological replicates.

We did not exclude any animals.

In the text, we have chosen to present means +/- SEM, while in the figures we have chosen to present box plots, which are useful for visualizing the distribution of the data across its quartiles.

The list and versions of softwares, patches used for scRNA-Seq analysis will be provided upon request.

Statistical analyses were performed using GraphPad Prism 8 and Excel.
<table><caption>KEY RESOURCES TABLE</caption><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse IgG1 anti hCD90-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1893U</td></tr><tr><td>Mouse IgG1 anti hCD109-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A08933</td></tr><tr><td>Mouse IgG1 anti hCD34-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2648U</td></tr><tr><td>Mouse IgG1 anti hCD34-APC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2472</td></tr><tr><td>Mouse IgG1 anti hCD117-PC5.5</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A66333</td></tr><tr><td>Mouse IgG1 anti hCD38-PC7</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A54189</td></tr><tr><td>Mouse IgG1 anti hCD3-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07747</td></tr><tr><td>Mouse IgG1 anti hCD4-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07750</td></tr><tr><td>Mouse IgG1 anti hCD8-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07757</td></tr><tr><td>Mouse IgG1 anti hCD19-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07768</td></tr><tr><td>Mouse IgG1 anti hIgM-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>B30655</td></tr><tr><td>Mouse IgG1 anti hTCR αβ-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>B49177</td></tr><tr><td>Mouse IgG1 anti hTCR γδ-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1571U</td></tr><tr><td>Mouse IgG1 anti hCD43-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM3264U</td></tr><tr><td>Mouse IgG1 anti hCD43-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A32560</td></tr><tr><td>Mouse IgG1 anti hCD45-KRO</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A96416</td></tr><tr><td>Mouse IgG1 anti hCD45-PC5.5</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A62835</td></tr><tr><td>Mouse IgG1 anti hCD14-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM0645U</td></tr><tr><td>Mouse IgG1 anti hCD33 APC-A750</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A70200</td></tr><tr><td>Mouse IgG1 anti hCD15-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1954U</td></tr><tr><td>Mouse IgG1 anti hCD13-ECD</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A33097</td></tr></tbody></table>
<table><caption>KEY RESOURCES TABLE</caption><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse IgG1 anti hCD90-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1893U</td></tr><tr><td>Mouse IgG1 anti hCD109-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A08933</td></tr><tr><td>Mouse IgG1 anti hCD34-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2648U</td></tr><tr><td>Mouse IgG1 anti hCD34-APC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2472</td></tr><tr><td>Mouse IgG1 anti hCD117-PC5.5</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A66333</td></tr><tr><td>Mouse IgG1 anti hCD38-PC7</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A54189</td></tr><tr><td>Mouse IgG1 anti hCD3-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07747</td></tr><tr><td>Mouse IgG1 anti hCD4-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07750</td></tr><tr><td>Mouse IgG1 anti hCD8-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07757</td></tr><tr><td>Mouse IgG1 anti hCD19-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07768</td></tr><tr><td>Mouse IgG1 anti hIgM-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>B30655</td></tr><tr><td>Mouse IgG1 anti hTCR αβ-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>B49177</td></tr><tr><td>Mouse IgG1 anti hTCR γδ-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1571U</td></tr><tr><td>Mouse IgG1 anti hCD43-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM3264U</td></tr><tr><td>Mouse IgG1 anti hCD43-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A32560</td></tr><tr><td>Mouse IgG1 anti hCD45-KRO</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A96416</td></tr><tr><td>Mouse IgG1 anti hCD45-PC5.5</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A62835</td></tr><tr><td>Mouse IgG1 anti hCD14-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM0645U</td></tr><tr><td>Mouse IgG1 anti hCD33 APC-A750</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A70200</td></tr><tr><td>Mouse IgG1 anti hCD15-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM1954U</td></tr><tr><td>Mouse IgG1 anti hCD13-ECD</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A33097</td></tr></tbody></table>
<table><tr><td>Mouse IgG1 anti hCD235a-FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2212</td></tr><tr><td>Mouse IgG1 anti hCD71-APC-A750</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A89313</td></tr><tr><td>Mouse IgG1 anti hCD146-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07483</td></tr><tr><td>Mouse IgG1 anti hCD309-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A64615</td></tr><tr><td>Mouse IgG1 anti hCD31-PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM02409</td></tr><tr><td>Mouse IgG1 anti hHLA-DR-PB</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A74781</td></tr><tr><td>Mouse IgG1 anti FITC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07795</td></tr><tr><td>Mouse IgG1 anti PE</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07796</td></tr><tr><td>Mouse IgG1 anti ECD</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A07797</td></tr><tr><td>Mouse IgG1 anti PC5.5</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A62833</td></tr><tr><td>Mouse IgG1 anti PC7</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>737662</td></tr><tr><td>Mouse IgG1 anti PB</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A74764</td></tr><tr><td>Mouse IgG1 anti APC A750</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>A79393</td></tr><tr><td>Mouse IgG1 anti APC</td><td>Beckman Coulter antibodies (Brea, USA)</td><td>IM2475</td></tr><tr><td>monoclonal mouse anti-alpha SMA</td><td>Sigma-Aldrich</td><td>A2547</td></tr><tr><td>monoclonal rabbit anti-NG2</td><td>Cell Signaling Technology</td><td>E3B3G</td></tr><tr><td>monoclonal rabbit anti-VECAD</td><td>Cell Signaling Technology</td><td>D87F2</td></tr><tr><td>monoclonal mouse anti-CD31</td><td>Cell Signaling Technology</td><td>89C2</td></tr><tr><td>monoclonal rabbit anti-RUNX1.</td><td>Abcam</td><td>92336</td></tr><tr><td>Goat anti-Mouse IgG1 Cross-Adsorbed, Alexa Fluor™488</td><td>Invitrogen</td><td>A-21121</td></tr><tr><td>Alexa Fluor® 647-AffiniPure</td><td>Jackson</td><td>711-605-152</td></tr></table>
<table>
  <tr>
    <td>Mouse IgG1 anti hCD235a-FITC</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>IM2212</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti hCD71-APC-A750</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A89313</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti hCD146-PE</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A07483</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti hCD309-PE</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A64615</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti hCD31-PE</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>IM02409</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti hHLA-DR-PB</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A74781</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti FITC</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A07795</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti PE</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A07796</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti ECD</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A07797</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti PC5.5</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A62833</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti PC7</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>737662</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti PB</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A74764</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti APC A750</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>A79393</td>
  </tr>
  <tr>
    <td>Mouse IgG1 anti APC</td>
    <td>Beckman Coulter antibodies (Brea, USA)</td>
    <td>IM2475</td>
  </tr>
  <tr>
    <td>monoclonal mouse anti-alpha SMA</td>
    <td>Sigma-Aldrich</td>
    <td>A2547</td>
  </tr>
  <tr>
    <td>monoclonal rabbit anti-NG2</td>
    <td>Cell Signaling Technology</td>
    <td>E3B3G</td>
  </tr>
  <tr>
    <td>monoclonal rabbit anti-VECAD</td>
    <td>Cell Signaling Technology</td>
    <td>D87F2</td>
  </tr>
  <tr>
    <td>monoclonal mouse anti-CD31</td>
    <td>Cell Signaling Technology</td>
    <td>89C2</td>
  </tr>
  <tr>
    <td>monoclonal rabbit anti-RUNX1.</td>
    <td>Abcam</td>
    <td>92336</td>
  </tr>
  <tr>
    <td>Goat anti-Mouse IgG1 Cross-Adsorbed, Alexa Fluor™488</td>
    <td>Invitrogen</td>
    <td>A-21121</td>
  </tr>
  <tr>
    <td>Alexa Fluor® 647-AffiniPure</td>
    <td>Jackson</td>
    <td>711-605-152</td>
  </tr>
</table>
<table><tr><td>Donkey Anti-Rabbit IgG (H+L)</td><td></td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>8961</td></tr><tr><td>Mouse IgG1 hCD3</td><td>Beckman Coulter</td><td>IM1280</td></tr><tr><td>Mouse IgG1 hCD28</td><td>Beckman Coulter</td><td>IM1376</td></tr><tr><td>Mouse IgGM anti SSEA1-FITC</td><td>BD Biosciences</td><td>560127</td></tr><tr><td>Mouse IgG3, κ anti-SSEA4-PE</td><td>BD Biosciences</td><td>560128</td></tr><tr><td>FITC Mouse IgM, κ Isotype Control RUO</td><td>BD Biosciences</td><td>553474</td></tr><tr><td>PE Mouse IgG3, κ Isotype Control RUO</td><td>BD Biosciences</td><td>559926</td></tr></table>

Chemicals, Peptides, and Recombinant Proteins

<table><tr><td>IMDM</td><td>Sigma-Aldrich</td><td>I3390</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td></tr><tr><td>Heparin</td><td>Choay</td><td>34869</td></tr><tr><td>Pool plasma AB</td><td>EFS</td><td></td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich</td><td>T4132</td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030081</td></tr><tr><td>SCF</td><td>MILTENYI</td><td>130-096-695</td></tr><tr><td>TPO</td><td>MILTENYI</td><td>130-094-013</td></tr><tr><td>Flt3I</td><td>MILTENYI</td><td>130-096-479</td></tr><tr><td>BMP4</td><td>MILTENYI</td><td>130-111-166</td></tr><tr><td>VEGF</td><td>MILTENYI</td><td>130-109-386</td></tr><tr><td>IL3</td><td>MILTENYI</td><td>130-095-068</td></tr><tr><td>IL6</td><td>MILTENYI</td><td>130-093-933</td></tr><tr><td>IL1 beta</td><td>MILTENYI</td><td>130-093-897</td></tr><tr><td>GCSF</td><td>MILTENYI</td><td>130-093-861</td></tr><tr><td>IGF1</td><td>MILTENYI</td><td>130-093-886</td></tr><tr><td>Matrigel hESC qualified</td><td>Corning</td><td>354277</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>C11330500BT</td></tr><tr><td>mTeSR™ Plus</td><td>STEMCELL Technologies</td><td>05825</td></tr><tr><td>ReLeSR™</td><td>STEMCELL Technologies</td><td>100-0484</td></tr><tr><td>D-PBS (Without Ca++ and Mg++)</td><td>Gibco</td><td>14190144</td></tr><tr><td>HSA</td><td>LFB</td><td>VIALEBEX</td></tr><tr><td>Methocult</td><td>Stemcell technologies</td><td>H4100</td></tr></table>
<table><tr><td>Donkey Anti-Rabbit IgG (H+L)</td><td></td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>8961</td></tr><tr><td>Mouse IgG1 hCD3</td><td>Beckman Coulter</td><td>IM1280</td></tr><tr><td>Mouse IgG1 hCD28</td><td>Beckman Coulter</td><td>IM1376</td></tr><tr><td>Mouse IgGM anti SSEA1-FITC</td><td>BD Biosciences</td><td>560127</td></tr><tr><td>Mouse IgG3, κ anti-SSEA4-PE</td><td>BD Biosciences</td><td>560128</td></tr><tr><td>FITC Mouse IgM, κ Isotype Control RUO</td><td>BD Biosciences</td><td>553474</td></tr><tr><td>PE Mouse IgG3, κ Isotype Control RUO</td><td>BD Biosciences</td><td>559926</td></tr></table>

## Chemicals, Peptides, and Recombinant Proteins

<table><tr><td>IMDM</td><td>Sigma-Aldrich</td><td>I3390</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td></tr><tr><td>Heparin</td><td>Choay</td><td>34869</td></tr><tr><td>Pool plasma AB</td><td>EFS</td><td></td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich</td><td>T4132</td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030081</td></tr><tr><td>SCF</td><td>MILTENYI</td><td>130-096-695</td></tr><tr><td>TPO</td><td>MILTENYI</td><td>130-094-013</td></tr><tr><td>Flt3I</td><td>MILTENYI</td><td>130-096-479</td></tr><tr><td>BMP4</td><td>MILTENYI</td><td>130-111-166</td></tr><tr><td>VEGF</td><td>MILTENYI</td><td>130-109-386</td></tr><tr><td>IL3</td><td>MILTENYI</td><td>130-095-068</td></tr><tr><td>IL6</td><td>MILTENYI</td><td>130-093-933</td></tr><tr><td>IL1 beta</td><td>MILTENYI</td><td>130-093-897</td></tr><tr><td>GCSF</td><td>MILTENYI</td><td>130-093-861</td></tr><tr><td>IGF1</td><td>MILTENYI</td><td>130-093-886</td></tr><tr><td>Matrigel hESC qualified</td><td>Corning</td><td>354277</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>C11330500BT</td></tr><tr><td>mTeSR™ Plus</td><td>STEMCELL Technologies</td><td>05825</td></tr><tr><td>ReLeSR™</td><td>STEMCELL Technologies</td><td>100-0484</td></tr><tr><td>D-PBS (Without Ca++ and Mg++)</td><td>Gibco</td><td>14190144</td></tr><tr><td>HSA</td><td>LFB</td><td>VIALEBEX</td></tr><tr><td>Methocult</td><td>Stemcell technologies</td><td>H4100</td></tr></table>
<table><tr><td>Collagenase B</td><td>ROCHE</td><td>11088815001</td></tr><tr><td>Growth factor-reduced Matrigel</td><td>Corning</td><td>354230</td></tr><tr><td>EBM-2 Medium</td><td>Lonza, Walkersville, USA</td><td>CC-3156</td></tr></table>

## Critical Commercial Assays

<table><tr><td>CellTrace™ CFSE</td><td>Thermofisher Scientific</td><td>C34570</td></tr><tr><td>Chromium Next GEM Single Cell 3' LT Kit v3.1</td><td>10X Genomics</td><td>1000325</td></tr></table>

## Deposited Data

<table><tr><td>Sequencing data supporting the findings of this study</td><td>Gene Expression Omnibus (GEO)</td><td>GSE224081</td></tr><tr><td>Crosse et al. sequencing data</td><td>GEO</td><td>GSE151877</td></tr><tr><td>BM human dataset</td><td>https://panglaodb.se/view_data.php?sra=SRA779509&srs=SRS3805267</td><td>SRR7881423</td></tr><tr><td>BM mouse dataset</td><td>https://panglaodb.se/view_data.php?sra=SRA653146&srs=SRS3044246</td><td>SRR6835854</td></tr></table>

## Experimental Models: Cell Lines

<table><tr><td>PCI-CAU hiPSC (aka internally PC045 RESCUE)</td><td>Phenocell</td><td></td></tr><tr><td>PCI-1429 hiPSC</td><td>Phenocell</td><td></td></tr><tr><td>FD136-25 hiPSC</td><td>Viville Lab</td><td>Lapillonne H et al. Haematologica. 2010 Oct;95(10):1651-9.</td></tr><tr><td>IMR90-16 hiPSC</td><td>Lapillonne et al., 2010</td><td>Lapillonne H et al. Haematologica. 2010 Oct;95(10):1651-9.</td></tr><tr><td>LAM01.005 hiPS</td><td>IPS CDTC core facility</td><td></td></tr><tr><td>LAM02.002 hiPS</td><td>IPS CDTC core facility</td><td></td></tr><tr><td>Mouse Stromal cells MS5</td><td>DSMZ</td><td>ACC 441</td></tr></table>

## Experimental Models: Organisms/Strains

<table><tr><td>mouse : NOD/SCID-LtSz-scid/scid (NOD/SCID)</td><td>Charles River Laboratories, L'Abresle, France</td><td>394</td></tr><tr><td>mouse : NOD.Cg-PrkdcscidII2rgtm1Wjl/SzJ (NSG)</td><td>Charles River Laboratories, L'Abresle, France</td><td>614</td></tr></table>

## Oligonucleotides

<table><tr><td>TaqMan™ gene expression assays Human C-MYB</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00193527_m1</td></tr></table>
<table><tr><td>Collagenase B</td><td>ROCHE</td><td>11088815001</td></tr><tr><td>Growth factor-reduced Matrigel</td><td>Corning</td><td>354230</td></tr><tr><td>EBM-2 Medium</td><td>Lonza, Walkersville, USA</td><td>CC-3156</td></tr></table>

## Critical Commercial Assays

<table><tr><td>CellTrace™ CFSE</td><td>Thermofisher Scientific</td><td>C34570</td></tr><tr><td>Chromium Next GEM Single Cell 3' LT Kit v3.1</td><td>10X Genomics</td><td>1000325</td></tr></table>

## Deposited Data

<table><tr><td>Sequencing data supporting the findings of this study</td><td>Gene Expression Omnibus (GEO)</td><td>GSE224081</td></tr><tr><td>Crosse et al. sequencing data</td><td>GEO</td><td>GSE151877</td></tr><tr><td>BM human dataset</td><td>https://panglaodb.se/view_data.php?sra=SRA779509&srs=SRS3805267</td><td>SRR7881423</td></tr><tr><td>BM mouse dataset</td><td>https://panglaodb.se/view_data.php?sra=SRA653146&srs=SRS3044246</td><td>SRR6835854</td></tr></table>

## Experimental Models: Cell Lines

<table><tr><td>PCI-CAU hiPSC (aka internally PC045 RESCUE)</td><td>Phenocell</td><td></td></tr><tr><td>PCI-1429 hiPSC</td><td>Phenocell</td><td></td></tr><tr><td>FD136-25 hiPSC</td><td>Viville Lab</td><td>Lapillonne H et al. Haematologica. 2010 Oct;95(10):1651-9.</td></tr><tr><td>IMR90-16 hiPSC</td><td>Lapillonne et al., 2010</td><td>Lapillonne H et al. Haematologica. 2010 Oct;95(10):1651-9.</td></tr><tr><td>LAM01.005 hiPS</td><td>IPS CDTC core facility</td><td></td></tr><tr><td>LAM02.002 hiPS</td><td>IPS CDTC core facility</td><td></td></tr><tr><td>Mouse Stromal cells MS5</td><td>DSMZ</td><td>ACC 441</td></tr></table>

## Experimental Models: Organisms/Strains

<table><tr><td>mouse : NOD/SCID-LtSz-scid/scid (NOD/SCID)</td><td>Charles River Laboratories, L'Abresle, France</td><td>394</td></tr><tr><td>mouse : NOD.Cg-PrkdcscidII2rgtm1Wjl/SzJ (NSG)</td><td>Charles River Laboratories, L'Abresle, France</td><td>614</td></tr></table>

## Oligonucleotides

<table><tr><td>TaqMan™ gene expression assays Human C-MYB</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00193527_m1</td></tr></table>
<table><tr><td>TaqMan™ gene expression assays Human TCF7</td><td>Thermofisher Scientific</td><td>4331182<br>Hs01556515_m1</td></tr><tr><td>TaqMan™ gene expression assays Human RUNX1</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00231079_m1</td></tr><tr><td>TaqMan™ gene expression assays Human HPRT1</td><td>Thermofisher Scientific</td><td>4331182<br>Hs01003267_m1</td></tr><tr><td>TaqMan™ gene expression assays Human CD19</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00174333_m1</td></tr><tr><td>TaqMan™ gene expression assays Human HBG</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00361131_g1</td></tr><tr><td>TaqMan™ gene expression assays Human HBB</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00758889_s1</td></tr><tr><td>TaqMan™ gene expression assays Human HBE</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00362216_m1</td></tr><tr><td>TaqMan™ gene expression assay Human GAPDH</td><td>Thermofisher Scientific</td><td>4331182<br>Hs02758991_g1</td></tr><tr><td>TaqMan™ array Human hematopoiesis</td><td>Thermofisher Scientific</td><td>4418803</td></tr></table>

## Software and Algorithms

<table><tr><td>CellRanger 6.1.2.</td><td>10X Genomics</td><td></td></tr><tr><td>SCTransform</td><td>github</td><td>https://github.com/satijalab/sctransform</td></tr><tr><td>SoupX</td><td>Young, M.D., Behjati, S. Gigascience (2020) doi: 10.1093/gigascience/giaa151 <sup>61</sup></td><td>https://github.com/constantAmateur/SoupX</td></tr><tr><td>scHCL mapping</td><td><u>Han, X. et al. Construction of a human cell landscape at singlecell level. Nature</u><br><u>https://doi.org/10.1038/s41586-020-2157-4 (2020)</u>.<sup>62</sup></td><td>https://github.com/ggjlab/scHCL</td></tr><tr><td>sc MCA mapping</td><td>Han et al., 2018, Cell. DOI:<u>https://doi.org/10.1016/j.cell.2018.02.001</u>.<sup>63</sup></td><td>https://github.com/ggjlab/scMCA</td></tr><tr><td>Seurat</td><td><sup>64</sup></td><td>https://github.com/satijalab/seurat</td></tr><tr><td>JMP</td><td>SAS, Institute Inc., Cary, USA</td><td>8.0</td></tr><tr><td>PRISM</td><td>GRAPHPAD</td><td>8.0</td></tr><tr><td>Flowjo</td><td>BD</td><td>X</td></tr></table>
<table><tr><td>TaqMan™ gene expression assays Human TCF7</td><td>Thermofisher Scientific</td><td>4331182<br>Hs01556515_m1</td></tr><tr><td>TaqMan™ gene expression assays Human RUNX1</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00231079_m1</td></tr><tr><td>TaqMan™ gene expression assays Human HPRT1</td><td>Thermofisher Scientific</td><td>4331182<br>Hs01003267_m1</td></tr><tr><td>TaqMan™ gene expression assays Human CD19</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00174333_m1</td></tr><tr><td>TaqMan™ gene expression assays Human HBG</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00361131_g1</td></tr><tr><td>TaqMan™ gene expression assays Human HBB</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00758889_s1</td></tr><tr><td>TaqMan™ gene expression assays Human HBE</td><td>Thermofisher Scientific</td><td>4331182<br>Hs00362216_m1</td></tr><tr><td>TaqMan™ gene expression assay Human GAPDH</td><td>Thermofisher Scientific</td><td>4331182<br>Hs02758991_g1</td></tr><tr><td>TaqMan™ array Human hematopoiesis</td><td>Thermofisher Scientific</td><td>4418803</td></tr></table>

## Software and Algorithms

<table><tr><td>CellRanger 6.1.2.</td><td>10X Genomics</td><td></td></tr><tr><td>SCTransform</td><td>github</td><td>https://github.com/satijalab/sctransform</td></tr><tr><td>SoupX</td><td>Young, M.D., Behjati, S. Gigascience (2020) doi: 10.1093/gigascience/giaa151 <sup>61</sup></td><td>https://github.com/constantAmateur/SoupX</td></tr><tr><td>scHCL mapping</td><td>Han, X. et al. Construction of a human cell landscape at singlecell level. Nature <a href="https://doi.org/10.1038/s41586-020-2157-4">https://doi.org/10.1038/s41586-020-2157-4 (2020)</a>.<sup>62</sup></td><td>https://github.com/ggjlab/scHCL</td></tr><tr><td>sc MCA mapping</td><td>Han et al., 2018, Cell. DOI:<a href="https://doi.org/10.1016/j.cell.2018.02.001">https://doi.org/10.1016/j.cell.2018.02.001</a>.<sup>63</sup></td><td>https://github.com/ggjlab/scMCA</td></tr><tr><td>Seurat</td><td><sup>64</sup></td><td>https://github.com/satijalab/seurat</td></tr><tr><td>JMP</td><td>SAS, Institute Inc., Cary, USA</td><td>8.0</td></tr><tr><td>PRISM</td><td>GRAPHPAD</td><td>8.0</td></tr><tr><td>Flowjo</td><td>BD</td><td>X</td></tr></table>
## LITTERATURE MINING

<figure><img src="image_3.png" /></figure>

## INPUT VARIABLES

Concentrations of growth factors involved in mesoderm commitment: **BMP4, VEGF**
Concentrations of cytokines involved in hematopoietic commitment

**SCF, TPO, FLT3, IL3, IL6, IL1, GCSF, IGF**

## OUTPUT VARIABLES

Cell amplification

Cell characterization by flow cytometry
**CD34, CD117, CD43, CD45, CD13, CD33, CD38, CD90, CD109, HLA-DR**

Long Term Culture-Initiating Cell (LTC-IC) frequency

## STATISTICAL STEPS

<figure><img src="image_4.png" /></figure>

## CUSTOM DESIGN

16 experiments displaying statistically different concentrations of cytokines and growth factors

Cell amplification : 16 results

Flow cytometry : 144 results

LTC-IC frequency : 16 results

<figure><img src="image_5.png" /></figure>

### Mathematical modeling of the input/output variable interactions

Three combinations of cytokines were selected to obtain highly amplifying cells harboring **CD34, CD117, CD43, CD45** hematopoietic commitment

### Flow cytometric analysis of D17 EBs

Tested on two different hiPSC lines, FD136-25 and IMR90

<figure><img src="image_6.png" /></figure>

### NOD SCID primary recipients

<figure><img src="image_7.png" /></figure>

## RESULTS

<figure><img src="image_8.png" /></figure>
LITTERATURE
MINING

<figure><img src="image_3.png" /></figure>

INPUT VARIABLES

Concentrations of growth factors involved
in mesoderm commitment : **BMP4**, **VEGF**
Concentrations of cytokines involved in
hematopoietic commitment

SCF, TPO, FLT3, IL3, IL6, IL1, GCSF,
IGF

OUTPUT VARIABLES

Cell amplification

Cell characterization by flow cytometry
CD34, CD117, CD43, CD45, CD13,
CD33, CD38, CD90, CD109, HLA-DR

Long Term Culture-Initiating Cell
(LTC-IC) frequency

CUSTOM DESIGN

16 experiments displaying statistically different concentrations of cytokines and growth factors

Cell amplification : 16 results

Flow cytometry : 144 results

LTC-IC frequency : 16 results

<figure><img src="image_4.png" /></figure>

Mathematical modeling of the input/output variable interactions

Three combinations of cytokines were selected to obtain highly amplifying cells
harboring CD34, CD117, CD43, CD45 hematopoietic commitment

Flow cytometric analysis of D17 EBs

Tested on two different hiPSC lines, FD136-25 and IMR90

<figure><img src="image_5.png" /></figure>

NOD SCID primary recipients

<figure><img src="image_6.png" /></figure>

STATISTICAL
STEPS

<figure><img src="image_7.png" /></figure>

RESULTS

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 2</figcaption></figure>

NSG primary recipient, BM, 20 weeks

<figure><img src="image_10.png" /></figure>

### F D17 EB cells. Multilineage analysis. BM primary transplant. Representative sample

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

### G D17 EB cells. Multilineage analysis. Thymus primary transplant. Representative sample

<figure><img src="image_13.png" /></figure>

### H Multilineage analysis on primary recipients

<figure><img src="image_14.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure 2</figcaption></figure>

NSG primary recipient, BM, 20 weeks

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_15.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>Figure 4</figcaption></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

E

F

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<table><thead><tr><th></th><th>Lower</th><th>Estimate</th><th>Upper</th></tr></thead><tbody><tr><td>D17</td><td>31715</td><td>15700</td><td>7773</td></tr></tbody></table>
NSG secondary recipient, BM, 20 weeks

<figure><img src="image_17.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_19.png" /></figure>

Hematopoietic engraftment per hiPSC line

<caption>D</caption>

Multilineage analysis per hiPSC line

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>E</figcaption></figure>

D17 EB cells. NSG secondary recipient, BM 20 weeks. Representative sample

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_25.png" /></figure>
NSG secondary recipient, BM, 20 weeks

<figure><img src="image_21.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_23.png" /></figure>

Hematopoietic engraftment per hiPSC line

<caption>D</caption>

Multilineage analysis per hiPSC line

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>E</figcaption></figure>

D17 EB cells. NSG secondary recipient, BM 20 weeks. Representative sample

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_29.png" /></figure>
<figure><img src="image_26.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>Figure 7</figcaption></figure>
SUPPLEMENTAL FIGURES AND TABLES (PIAU ET AL.,)

SUPPLEMENTAL FIGURES
SUPPLEMENTAL FIGURES AND TABLES (PIAU ET AL.,)

SUPPLEMENTAL FIGURES
<caption>A</caption>

PCI-CAU D17 EB processed by an ATMP platform
NSG primary recipients
8 weeks

<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

PCI-CAU D17 EB processed by an ATMP platform
NSG primary recipients
12 weeks

<figure><img src="image_29.png" /><figcaption>C</figcaption></figure>

PCI-CAU
NSG primary recipients
Kinetic of engraftment

<figure><img src="image_30.png" /></figure>

Multilineage in saline-transplanted control

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

CD45⁺CD34⁺CD38⁺ and CD45⁺CD34⁺CD38⁺ populations in BM first recipient

<figure><img src="image_33.png" /></figure>

Peripheral blood. Multilineage analysis

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

Thymus

Spleen

Thymus and spleen multilineage analysis

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>
<caption>A</caption>

PCI-CAU D17 EB processed by an ATMP platform
NSG primary recipients
8 weeks

<figure><img src="image_32.png" /><figcaption>B</figcaption></figure>

PCI-CAU D17 EB processed by an ATMP platform
NSG primary recipients
12 weeks

<figure><img src="image_33.png" /><figcaption>C</figcaption></figure>

PCI-CAU
NSG primary recipients
Kinetic of engraftment

<figure><img src="image_34.png" /></figure>

Multilineage in saline-transplanted control

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

CD45⁺CD34⁺CD38⁺ and CD45⁺CD34⁺CD38⁻ populations in BM first recipient

<figure><img src="image_37.png" /></figure>

Peripheral blood. Multilineage analysis

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

Thymus and spleen multilineage analysis

<figure><img src="image_41.png" /></figure>

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**Supplementary Figure 1 is related to Figure 2.**

**In vivo engraftment of D17 human EB cells in primary NSG mice.**

(A-B) D17 EB cells from PCi-CAU, prepared by the Atlantic Bio GMP platform (Nantes, France), (A) multilineage analysis at 8 weeks (5 mice, n=1). (B) at 12 weeks (n=5, 1 experiment). Post grafts. FACS results are expressed as % of hCD34+, hCD43+, and hCD45+. Wilcoxon Mann-Whitney test. Data are mean±SD or SEM depending on the number of independent experiments

(C) Kinetics of cell grafting at 8, 12, and 20 weeks (n=5 mice, 5 mice, 1 experiment for 8 and 12 weeks respectively and, n=59 mice, 14 independent experiments) post-transplantation resulting from pooling of in-house and Atlantic Bio GMP platform results. FACS results are expressed as % of hCD45+.

(D) human Multilineage engraftment. Flow cytometric analysis of BM. Representative control (saline-injected) primary recipients at 20 weeks (n=16, 8 independent experiments) showing Myeloid (M), B, T and Pro-erythroid cells (Pro-E). hCD45+ population was analyzed for expression of hCD14/hCD15 (monocytes/macrophages), hCD19/hIgM (B cells), and hCD3 (T cells). hCD71/hCD235a (ProE) analysis was performed on whole BM cells.

(E) Representative primary recipients transplanted with 4.10⁵ CD34+ CB cells (n=13 mice, 3 independent experiments). Same analysis as in (D).

(F) Representative FACS analysis of D17 grafts BM for human and mouse CD45 expression.

(G) Representative FACS analysis of D17 grafts BM for human CD34 and CD38 expression. Numbers indicate mean ± SEM of 14 mice analyzed. (n=14, 2 independent experiments).

(H) Representation of hCD45+ hCD34+ hCD38+ and hCD45+ hCD34+ hCD38- populations. (n=14, 2 independent experiments).

(I-K) Analysis of NSG peripheral blood in control (I, n=16, 8 independent experiments), D17EB (J, n=59, 14 independent experiments) and CD34+ CB cell (K, n=13, 3 independent experiments)-transplanted mice. Primary recipients at 20 weeks. Myeloid (hCD11B/CD14) and lymphoid (CD19/IgM and CD4/CD8) lineages in peripheral blood. Analyzes were performed on human CD45+ cells, except for saline-transplanted recipients.

(L-O) Analysis of NSG thymus (J, K) and spleen (L, M). Primary recipients at 20 weeks.

(L-M) Representative thymuses from NSG mice transplanted with D17 EB cells (H, 59 mice, n=14) or CD34+ CB cells (13 mice, n=3). hTCR alpha/beta and gamma/delta flow cytometric analysis. The CD3-gated thymus cells had a large amount of TCR alpha-beta cells, which was very similar to the amount on the CD34+ CB cells.

(N-O) Representative NSG spleen. hTCR beta and hCD19 flow cytometric analysis. (K) D17 EB cell-transplanted NSG (n=59, 14 independent experiments). (L) CD34+ CB cells-transplanted NSG (13 mice, n=3). The hCD45+-gated splenocytes exhibited a B- or T-lymphoid phenotype, with similar ratios as the CD34+ CB cells.

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 1 is related to Figure 2.**

**In vivo engraftment of D17 human EB cells in primary NSG mice.**

(A-B) D17 EB cells from PCi-CAU, prepared by the Atlantic Bio GMP platform (Nantes, France), (A) multilineage analysis at 8 weeks (5 mice, n=1). (B) at 12 weeks (n=5, 1 experiment). Post grafts. FACS results are expressed as % of hCD34+, hCD43+, and hCD45+. Wilcoxon Mann-Whitney test. Data are mean±SD or SEM depending on the number of independent experiments

(C) Kinetics of cell grafting at 8, 12, and 20 weeks (n=5 mice, 5 mice, 1 experiment for 8 and 12 weeks respectively and, n=59 mice, 14 independent experiments) post-transplantation resulting from pooling of in-house and Atlantic Bio GMP platform results. FACS results are expressed as % of hCD45+.

(D) human Multilineage engraftment. Flow cytometric analysis of BM. Representative control (saline-injected) primary recipients at 20 weeks (n=16, 8 independent experiments) showing Myeloid (M), B, T and Pro-erythroid cells (Pro-E). hCD45+ population was analyzed for expression of hCD14/hCD15 (monocytes/macrophages), hCD19/hIgM (B cells), and hCD3 (T cells). hCD71/hCD235a (ProE) analysis was performed on whole BM cells.

(E) Representative primary recipients transplanted with 4.10⁵ CD34+ CB cells (n=13 mice, 3 independent experiments). Same analysis as in (D).

(F) Representative FACS analysis of D17 grafts BM for human and mouse CD45 expression.

(G) Representative FACS analysis of D17 grafts BM for human CD34 and CD38 expression. Numbers indicate mean ± SEM of 14 mice analyzed. (n=14, 2 independent experiments).

(H) Representation of hCD45+ hCD34+ hCD38+ and hCD45+ hCD34+ hCD38- populations. (n=14, 2 independent experiments).

(I-K) Analysis of NSG peripheral blood in control (I, n=16, 8 independent experiments), D17EB (J, n=59, 14 independent experiments) and CD34+ CB cell (K, n=13, 3 independent experiments)-transplanted mice. Primary recipients at 20 weeks. Myeloid (hCD11B/CD14) and lymphoid (CD19/IgM and CD4/CD8) lineages in peripheral blood. Analyzes were performed on human CD45+ cells, except for saline-transplanted recipients.

(L-O) Analysis of NSG thymus (J, K) and spleen (L, M). Primary recipients at 20 weeks.

(L-M) Representative thymuses from NSG mice transplanted with D17 EB cells (H, 59 mice, n=14) or CD34+ CB cells (13 mice, n=3). hTCR alpha/beta and gamma/delta flow cytometric analysis. The CD3-gated thymus cells had a large amount of TCR alpha-beta cells, which was very similar to the amount on the CD34+ CB cells.

(N-O) Representative NSG spleen. hTCR beta and hCD19 flow cytometric analysis. (K) D17 EB cell-transplanted NSG (n=59, 14 independent experiments). (L) CD34+ CB cells-transplanted NSG (13 mice, n=3). The hCD45+-gated splenocytes exhibited a B- or T-lymphoid phenotype, with similar ratios as the CD34+ CB cells.

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