In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras

  1. Bingqiang Wen
  2. Guolun Wang
  3. Enhong Li
  4. Olena A Kolesnichenko
  5. Zhaowei Tu
  6. Senad Divanovic
  7. Tanya V Kalin
  8. Vladimir V Kalinichenko  Is a corresponding author
  1. Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, United States
  2. Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, United States
  3. Division of Immunobiology, Cincinnati Children's Hospital Medical Center, United States
  4. Department of Pediatrics, College of Medicine of the University of Cincinnati, United States
  5. Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, United States
  6. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, United States

Abstract

Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on fluorescence-activated cell sorting analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor–ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse–rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse–rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse–rat chimeras rescued lethally irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional BM was generated from mouse ESCs using rat embryos as ‘bioreactors’.

Editor's evaluation

This work convincingly establishes a chimeric blastocyst complementation assay as a "bioreactor" to study the differentiation of mouse embryonic stem cells into hematopoietic lineages. The elegance of the approach lies in the use of GFP+ mouse embryonic stem cells that are implanted into a rat blastocyst, thus allowing for the tracking and phenotyping of the mouse-derived GFP+ hematopoietic cells in the post-natal rat. This is an important contribution that will be of interest to researchers in developmental biology and hematopoiesis.

https://doi.org/10.7554/eLife.74018.sa0

Introduction

The bone marrow (BM) is a remarkably complex organ consisting of multiple mesenchymal, immune, endothelial, and neuronal cell types which together comprise a highly specialized microenvironment required to support c blood regeneration or hematopoiesis (Baccin et al., 2020; Baryawno et al., 2019; Rowe et al., 2016; Tikhonova et al., 2019; Vo and Daley, 2015). Hematopoiesis occurs in a stepwise manner and is initiated by a heterogeneous, multipotent, population of hematopoietic stem cells (HSCs), located at the apex of the hematopoietic differentiation tree. Long-term HSCs (LT-HSCs) remain quiescent to maintain their undifferentiated state within the BM niche. When necessary, LT-HSCs can either undergo differentiation or self-renewal, to maintain the HSC pool. Conversely, short-term HSCs (ST-HSCs) are restricted in their self-renewal capacity and primed for differentiation into multipotent progenitors (MPPs), initiating the process of blood cell development. MPPs further differentiate into common myeloid progenitors (CMPs), lymphoid-primed multipotent progenitors (LMPPs), and common lymphoid progenitors (CLPs) that become increasingly lineage restricted with subsequent cell divisions, ultimately yielding all mature blood cell types (Haas et al., 2018). The complexities of the hematopoietic system have been studied extensively in vitro, utilizing paired-daughter and colony-forming unit (CFU) assays (Rowe et al., 2016; Vo and Daley, 2015). Fluorescence-activated cell sorting (FACS) has allowed for precise isolation and characterization of HSCs and progenitor populations based on cell surface markers. Classically, the most biologically relevant way to test HSC function remains to be through serial transplantation and hematopoietic reconstitution of irradiated recipient mice (Purton and Scadden, 2007; Rowe et al., 2016; Vo and Daley, 2015). Recent advances in single-cell RNA sequencing (scRNAseq) have made it possible to further explore heterogeneity of the BM niche (Baryawno et al., 2019; Tikhonova et al., 2019), and identify gene expression signatures of hematopoietic progenitor cells as they differentiate into mature blood cell types (Baccin et al., 2020; Nestorowa et al., 2016).

Generation of functional BM from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) promises to provide new therapeutic opportunities for hematologic and autoimmune disorders. However, this approach is limited by technical challenges to produce functional HSCs or the mixture of hematopoietic progenitors capable of replacing all mature blood cell types after cell transplantation. HSC-like cells have been generated from mouse and human ESCs and iPSCs using in vitro differentiation protocols (Amabile et al., 2013; Doulatov et al., 2013; Grigoriadis et al., 2010; Kitajima et al., 2011; Ledran et al., 2008; Sugimura et al., 2017; Vodyanik et al., 2006). Likewise, ESCs and iPSCs have been used to produce myeloid and lymphoid progenitor cells as well as differentiated hematopoietic cells, including neutrophils, monocytes, erythroid cells, and T and B lymphocytes (Doulatov et al., 2013; Elcheva et al., 2014; Galic et al., 2006; Kennedy et al., 2012; Montel-Hagen et al., 2019; Nafria et al., 2020; Vodyanik et al., 2005). When transplanted into irradiated animals, ESC/iPSC-derived hematopoietic progenitor cells undergo differentiation and engraft into the BM niche, providing an important source of renewal and regeneration for various blood cell lineages (Rowe et al., 2016; Sugimura et al., 2017; Vo and Daley, 2015). While ESC/iPSC-derived hematopoietic cells often express appropriate cell markers, gene expression and functional studies indicate significant differences between ESC/iPSC-derived cells and endogenous cells that have undergone normal morphogenesis in the BM (Lin et al., 2019; Lu et al., 2016; Sugimura et al., 2017).

In vivo differentiation of ESCs into multiple cell lineages can be achieved using blastocyst complementation, in which donor ESCs are injected into blastocysts of recipient animals to create chimeras. Fluorescently labeled ESCs undergo differentiation in recipient embryos that serve as ‘biological reactors’ by providing growth factors, hormones, and cellular niches to support ESC differentiation in the embryo. In mouse and rat apancreatic Pdx1−/− embryos, donor ESCs formed an entire pancreas in which both exocrine and endocrine cells were almost entirely derived from ESCs or iPSCs (Kobayashi et al., 2010; Yamaguchi et al., 2017). Mouse ESC/iPSC-derived β-cells from mouse–rat chimeras were fully differentiated and successfully rescued syngeneic diabetic mice (Yamaguchi et al., 2017). ESCs generated pancreatic cell lineages in apancreatic pigs (Matsunari et al., 2013), kidney in Sall1-deficient rats (Goto et al., 2019), endothelial cells in Flk1−/− mice (Hamanaka et al., 2018), lymphocytes in immunodeficient mice (Muthusamy et al., 2011), and neuronal progenitors in mice with forebrain-specific overexpression of diphtheria toxin (Chang et al., 2018). Recently, mouse ESCs were used to generate lung and thyroid tissues in embryos deficient for Fgf10, Nkx2-1, Fgfr2, or β-catenin (Kitahara et al., 2020; Mori et al., 2019; Wen et al., 2021). Using blastocyst complementation, mouse ESCs effectively produced hematopoietic cells in mice deficient for Kit or Flk1 (Hamanaka et al., 2018; Jansson and Larsson, 2010). ESC-derived endothelial progenitor cells from mouse–rat chimeras were indistinguishable from endogenous endothelial progenitor cells based on gene expression signatures and functional properties (Wang et al., 2021), indicating that ESC/iPSC-derived progenitors can be used for tissue regeneration (Bolte et al., 2020a; Bolte et al., 2018; Dharmadhikari et al., 2015; Kolesnichenko et al., 2021). While all these studies support the effectiveness of blastocyst complementation for differentiation of multiple cell types from ESCs/iPSCs in vivo, generation of functional BM from ESCs in interspecies chimeras has not yet been achieved.

Herein, we used blastocyst complementation to produce mouse BM in a rat. ESC-derived cells from multiple hematopoietic and stromal cell lineages were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells into lethally irradiated syngeneic mice prevented mortality and resulted in a long-term contribution to BM and mature blood cell types. Our data demonstrate that interspecies chimeras can be used as ‘bioreactors’ for in vivo differentiation and functional studies of ESC-derived BM hematopoietic and stromal cells.

Results

Generation of BM from pluripotent ESCs in interspecies mouse–rat chimeras

To determine whether mouse ESCs can differentiate into multiple hematopoietic cell lineages in the BM of a rat, blastocyst complementation was performed by injecting GFP-labeled mouse C57BL/6 ESCs (ESC-GFP) into rat SD blastocysts to create interspecies mouse–rat chimeras. Chimeric embryos were transferred into surrogate female rats for subsequent development in utero (Figure 1A). While mouse–rat chimeras were viable, they were smaller than age-matched rats (Figure 1B). Consistent with the presence of mouse ESC-derived cells (black) in the skin tissue (Wang et al., 2021), mixed black and white pigmentation distinguished the mouse–rat chimeras from juvenile rats (Figure 1B). The average body weight of mouse–rat chimeras was smaller than rats, but larger than mice of similar age (Figure 1C). ESC-derived cells were abundant in femur and tibia bones of the chimeras as evidenced by GFP fluorescence (Figure 1D). FACS analysis of BM cells obtained from juvenile mouse–rat chimeras revealed that the percentage of ESC-derived cells was 15–50% (Figure 1E, F). Thus, ESCs contribute to the BM of mouse–rat chimeras.

Figure 1 with 1 supplement see all
Mouse embryonic stem cells (ESCs) contribute to hematopoietic stem cells (HSCs) in the bone marrow (BM) of mouse–rat chimeras.

(A) Schematic shows blastocyst complementation of rat embryos with mouse ESCs to generate interspecies mouse–rat chimeras. GFP-labeled mouse ESCs (mESCs) were injected into rat blastocysts, which were implanted into surrogate rat females to undergo embryonic development in utero. Femur and tibia bones of the chimeras were used to obtain BM cells. (B) Photographs of mouse–rat chimeras are taken at postnatal (P) days P3, P8, P13, and P28. Mixed black and white pigmentation distinguishes the mouse–rat chimeras from juvenile rats and mice. (C) Weights of mouse–rat chimeras are shown at different time points and compared to rats and mice of similar ages. Chimeras are significantly smaller than rats, but larger than mice (n = 7–18 in each group), **p < 0.01, see also Source data 1. (D) Fluorescence microscopy shows GFP and bright-field images of femur and tibia bones from P4 rat, mouse, and mouse–rat chimera. (E) Fluorescence-activated cell sorting (FACS) analysis of mouse ESC-derived (GFP-positive) cells in the BM of P10 mouse–rat chimeras. Lineage-negative (Lin), LSK, short-term HSC (ST-HSC), and long-term HSC (LT-HSC) cell subsets were identified in the BM of mouse–rat chimeras (n = 10) and control mice (n = 8), see also Figure 1—figure supplement 1A, B. (F) Histograms show GFP fluorescence of BM cells from chimeras and control mice. (G–H) FACS analysis shows increased percentages of mouse LSKs and LT-HSCs in BM of mouse–rat chimeras (n = 10) compared to control mice (n = 8), **p < 0.01, N.S. indicates no significance. (I) FACS analysis shows increased numbers of HSCs (ST-HSCs + LT-HSCs) in BM of mouse rat chimeras (n = 10) compared to control mice (n = 8), **p < 0.01.

To identify ESC-derived HSCs, we used GFP fluorescence and mouse-specific antibodies recognizing multiple cell surface antigens (Figure 1E and Figure 1—figure supplement 1A, B). First, ESC-derived GFP+ BM cells were subdivided into lineage-positive (Lin+) and lineage-negative subpopulations (Lin) (Figure 1E and Figure 1—figure supplement 1A, B). The percentage of ESC-derived Lin cells in the BM of mouse–rat chimeras was similar to the percentage of Lin cells in the BM of age-matched C57BL/6 mice (Figure 1E, G). Next, we used Sca1 and CD117 (c-KIT) antibodies to identify LinSca1+c-KIT+ cells (LSKs) (Figure 1E). The percentage of LSKs was higher in the BM of mouse–rat chimeras compared to the control (Figure 1G). Based on cell surface expression of CD150 and CD48, the percentage of LT-HSCs among LSKs was also higher in mouse–rat chimeras (Figure 1E, H). While changes in ST-HSCs were not significant (Figure 1H), total numbers of HSCs (LT-HSCs + ST-HSCs) were higher in mouse–rat chimeras compared to mice of the same age (Figure 1I). Thus, mouse ESCs can differentiate into hematopoietic progenitor cells in the BM of mouse–rat chimeras.

Single-cell RNA sequencing identifies multiple subpopulations of ESC-derived hematopoietic cells in the BM of mouse–rat chimeras

To identify ESC-derived cells in the BM, single-cell RNAseq (the 10× Chromium platform) of FACS-sorted GFP+ BM cells was performed. Mouse ESC-derived cells from P10 mouse–rat chimeras were compared to ESC-derived cells from P10 mouse–mouse (control) chimeras, the latter of which were produced by complementing mouse blastocysts with mouse ESCs from the same ESC-GFP cell line. Based on GFP fluorescence, contribution of ESCs to BM cells in both chimeras was similar (Figure 2—figure supplement 1A, B). Since the numbers of HSCs and other hematopoietic progenitor cells in the BM are low compared to numbers of differentiated hematopoietic cells, we enriched for BM progenitor cell populations prior to single-cell RNA sequencing by combining 90% of FACS-sorted GFP+Lin cells and 10% of GFP+Lin+ cells in each experimental group. BM cells from 3 animals per group were combined prior to FACS sorting. Based on published gene expression signatures of mouse BM cells (Baccin et al., 2020), 11,326 cells from 14 major cell subtypes were identified: 5308 cells from control mouse–mouse chimeras and 6018 cells from mouse–rat chimeras. These include lymphoid, erythroid, myeloid, and neutrophil progenitors, Pro-B, Pre-B, B and T lymphocytes, megakaryocytes, dendritic cells, neutrophils, basophils/eosinophils, monocytes, and LMPP cells (Figure 2A and Figure 2—figure supplement 2A). Analysis of BM cells from mouse–rat and mouse–mouse chimeras demonstrated similar distributions of hematopoietic cell lineages derived from CMP and CLP (Figure 2A), indicating identical cell types in mouse–rat and control chimeras. For selected genes, we used violin plots to confirm cell specificity and expression levels of Ptprc (Cd45), Pclaf, Vpreb1, Tmpo, Ebf1, Ms4a4b, Vamp5, Elof1, Elane, Ms4a2, Siglech, Ngp, Clec4d, Ctss, and Ftl1-ps1 in the combined dataset (Figure 2—figure supplement 3). Markers of endothelial cells, adipocytes, osteocytes, and neuronal cells were undetectable in BM cell suspensions from both chimeras (Figure 2—figure supplement 2B). Percentages CLP-derived lymphoid progenitors, Pro-B, Pre-B, and B cells were lower in mouse–rat chimeras compared to the control (Figure 2A, B). In contrast, percentages of CMP-derived erythroid, myeloid and neutrophil progenitors, dendritic cells, and basophils/eosinophils were higher (Figure 2B). Monocytes and neutrophils were similar, whereas megakaryocytes were decreased in the BM of mouse–rat chimeras (Figure 2B). The percentage of LMPPs in mouse–rat chimeras was increased compared to the control (Figure 2A, B). HSCs, identified by coexpression of Kit, Ly6a(Sca1), and Flt3 mRNAs (Rowe et al., 2016; Vo and Daley, 2015), clustered together with myeloid and erythroid progenitors (Figure 2—figure supplement 4A, B). The number of ESC-derived HSCs was higher in BM of mouse–rat chimeras compared to the control (Figure 2—figure supplement 4C), findings consistent with FACS analysis (Figure 1H, I). Only 6 out of 6018 BM cells (0.1%) in mouse–rat chimeras contained both mouse and rat mRNA transcripts (Supplementary files 1 and 2), indicating that the fusion of mouse and rat BM cells is rare. Thus, although the cellular composition of ESC-derived hematopoietic BM cells was similar in mouse–rat and mouse–mouse chimeras, mouse–rat BM was enriched in HSCs, LMPPs, and CMP-derived erythroid, myeloid, and neutrophil progenitors.

Figure 2 with 7 supplements see all
Single-cell RNAseq analysis identifies embryonic stem cell (ESC)-derived hematopoietic cell lineages in the bone marrow (BM) of mouse–rat chimeras.

(A) Parallel dimension UMAP plots show identical hematopoietic cell clusters in the BM of mouse–mouse chimera (5308 cells) and mouse–rat chimera (6018 cells). ESC-derived BM cells were obtained from the BM of P10 chimeras using fluorescence-activated cell sorting (FACS) for GFP+ cells, see Figure 2—figure supplement 1A, B. Cells from n = 3 animals per group were pooled together prior to FACS sorting. Cell clusters were identified from single-cell RNAseq datasets using Uniform Manifold Approximation and Projection (UMAP) method, see also Figure 2—figure supplements 2A, B and 3. Hematopoietic stem cells (HSCs) were identified by coexpression of Kit, Ly6a (Sca1), and Flt3 (Flk2), see Figure 2—figure supplement 4. Heatmaps and linear regression analysis identified significant similarities in gene expression signatures of lymphoid and myeloid progenitor cells obtained from mouse–rat (R) and mouse–mouse chimeras (M), see Figure 2—figure supplement 5A, B and Figure 2—figure supplement 6A, B. Gene expression profiles of ESC-derived HSCs and lymphoid-primed multipotent progenitor cells are shown in Figure 2—figure supplement 7A, B. (B) Table shows percentages of cells in individual clusters in mouse–mouse and mouse–rat chimeras. Blue color indicates decreased percentages of cells in mouse–rat chimeras compared to mouse–mouse chimeras. Red color indicates increased percentages of cells in mouse–rat chimeras.

Single-cell RNA sequencing identifies close similarities in gene expression signatures between ESC-derived hematopoietic cells in mouse–rat and mouse–mouse chimeras

Comparison of gene expression signatures between mouse–rat and mouse–mouse chimeras revealed significant similarities among ESC-derived hematopoietic cell types. Lymphoid progenitors and pro-B cells isolated from mouse–rat and control chimeras expressed Mif, Rcsd1, and Tspan13, whereas pre-B cells expressed Hmgb2 and Pgls (Figure 2—figure supplement 5A). Cd79a and CD79b transcripts were detected in B cells of mouse–rat and control chimeras, whereas Cd3g and Lck were restricted to T cells (Figure 2—figure supplement 5A). Based on the correlation analysis, gene expression profiles of all lymphoid cell types were similar between mouse–rat and control chimeras (Figure 2—figure supplement 5B). Likewise, gene expression signatures of myeloid, erythroid, and neutrophil progenitors and their derivatives in the BM were similar in both experimental groups (Figure 2—figure supplement 6A, B). Furthermore, single-cell RNAseq identified close similarities in gene expression signatures of ESC-derived HSCs and LMPPs in both chimeras (Figure 2—figure supplement 7A, B). Thus, gene expression signatures of ESC-derived hematopoietic cells were similar in mouse–rat and control mouse–mouse chimeras.

Chimeric BM is enriched in mouse hematopoietic progenitor cells and rat chondrocytes

To examine the composition and origin of stromal cells in mouse–rat chimeras, we used an enzymatic digestion to obtain both hematopoietic and stromal cells from BM of P5 mouse–rat chimeras and compared them to BM cells of mice and rats of the same age. Flow sorting for GFP was performed to separate donor mouse cells (GFP+) and recipient rat cells (GFP) in the chimeric BM. BM from control P5 mice and rats was also FACS-sorted for GFP BM cells to ensure similar conditions of cell preparations prior to single-cell RNAseq. Based on published gene expression signatures (Baccin et al., 2020), 6375 mouse and 5495 rat cells were identified in the chimeras, which were compared to 6418 cells from control mice and 7016 cells from control rats. Similar hematopoietic and stromal cell clusters were present in BM of mice, rats, and mouse–rat chimeras (Figure 3A–C). These included stromal cell clusters (endothelial cells, fibroblasts, myofibroblasts, and chondrocytes) and hematopoietic cell clusters with various progenitor and differentiated hematopoietic cell types. Since we did not enrich BM cell populations for Lin cells, some rare BM cell subsets, such as HSCs, LMPPs, and dendritic cells, were not detected as separate cell clusters. Compared to normal BM from P5 mice, chimeric BM was enriched in mouse ESC-derived hematopoietic progenitor cells, such as myeloid, granulocyte, and erythroid progenitors, whereas mouse-derived B cell lineages were reduced (Figure 3A), findings consistent with single-cell RNAseq comparison of P10 BM from mouse–rat and mouse–mouse chimeras (Figure 2). The percentage of mouse endothelial cells was increased in mouse–rat BM, whereas the percentages of mouse chondrocytes and fibroblasts were reduced compared to mouse control (Figure 3A). In contrast, mouse–rat BM was enriched in rat-derived chondrocytes and fibroblasts, but the percentages of endothelial and most hematopoietic cells were reduced compared to age-matched rats (Figure 3B). Thus, mouse cells preferentially contributed to hematopoietic progenitors and endothelial cells, whereas rat cells contributed to the majority of chondrocytes and fibroblasts.

Figure 3 with 1 supplement see all
Single-cell RNAseq analysis shows increased percentages of embryonic stem cell (ESC)-derived hematopoietic progenitors and endothelial cells but decreased percentages of ESC-derived chondrocytes in the bone marrow (BM) of mouse–rat chimeras.

(A, B) Parallel dimension UMAP plots show identical hematopoietic and stromal cell clusters in the BM of P5 mice, rats, and mouse–rat chimeras. BM cells were obtained from P5 animals using an enzymatic digestion (n = 5 animals per group) and pooled prior to single-cell RNAseq. Cell clusters were identified from single-cell RNAseq datasets using Uniform Manifold Approximation and Projection (UMAP) method. Red color in the tables indicates increased percentages of cells in mouse–rat chimeras compared to either mice or rats of the same age. Blue color indicates decreased percentages of cells in mouse–rat chimeras. Gene expression signatures of mouse and rat hematopoietic and stromal cells are shown in Figure 3—figure supplement 1A–D. (C) A bar graph shows relative percentages of ESC-derived mouse cells (green) and endogenous rat cells (blue) in the BM of P5 mouse–rat chimeras.

Direct comparison of mouse and rat cells within chimeric BM demonstrated significant similarities between gene expression signatures of hematopoietic and stromal cell lineages (Figure 3—figure supplement 1A–D). To examine cell signaling between hematopoietic progenitors and stromal cells in BM of mouse–rat chimeras, we generated the map of potential ligand–receptor interactions using P5 single-cell RNAseq datasets. There were remarkable similarities in major receptor–ligand interactions between stromal and erythro-myeloid progenitor cells (EMPs) (Figure 4). Regardless of mouse and rat origins of BM cells, endothelial cells interacted with EMPs through the Cxcl12-Cxcr4 receptor–ligand signaling pair. The main signaling circuit between fibroblasts and EMPs was Lama2-Itga6, whereas chondrocytes signaled to EMPs through App-Itga6 and Comp-Cd47 pathways (Figure 4). Major receptor–ligand interactions between granulocyte–monocyte progenitor (GMP) cells and stromal cells were also similar in BM cells of mouse and rat origin (Figure 4—figure supplement 1). These include Cxcl12-Cxcr4 signaling between endothelial cells and GMPs, Col1a1-Cd44 signaling between fibroblasts and GMPs, and App-Il18rap signaling between chondrocytes and GMPs (Figure 4—figure supplement 1). Analysis of expression patterns for several ligands and their receptors revealed no obvious differences between mouse and rat cells (Figure 4—figure supplement 2). These results demonstrate that mouse and rat BM cells use similar signaling pathways between stromal and hematopoietic progenitor cells.

Figure 4 with 2 supplements see all
Single-cell RNAseq analysis shows remarkable similarities in major receptor–ligand interactions between erythro-myeloid progenitors and stromal cells of mouse and rat origins.

Bone marrow (BM) cells were obtained from P5 animals using an enzymatic digestion (n = 5 animals per group). Single-cell RNAseq was performed to identify BM stromal and erythro-myeloid progenitor cells (EMPs) based on gene expression signatures. The R package NicheNet was used to analyze the expression of ligands and receptors to identify intercellular communication patterns between EMPs and BM stromal cells. Receptor–ligand interactions between stromal and granulocyte–monocyte progenitor (GMP) cells are shown in Figure 4—figure supplement 1. Violin plots were used to identify expression of ligands and their receptors in hematopoietic and stromal BM cells, see Figure 4—figure supplement 2.

Mouse HSCs in mouse–rat chimeras develop earlier than rat HSCs

Fetal HSCs emerge from hemogenic endothelium in the aorta–gonad–mesonephros region and later undergo expansion in the embryonic liver (Gao et al., 2018; Weijts et al., 2021). To examine the development of HSCs in mouse–rat chimeras, mouse-derived (GFP+) and rat-derived (GFP) hemogenic endothelial cells were visualized in the dorsal aorta by colocalization of FLK1 with RUNX1 transcription factor (Figure 5A, B). At E11, mouse embryos were significantly larger than rat and mouse–rat chimeric embryos (Figure 5—figure supplement 1), consistent with previous studies demonstrating that the main stages of mouse embryonic development occur approximately 1.5 days faster compared to embryonic development in the rat (Farrington-Rock et al., 2008; Marcela et al., 2012; Takahashi and Osumi, 2005; Torres et al., 2008). Therefore, we compared E11 mouse embryos with E12.5 rat and chimeric embryos which were in similar developmental stages. In the dorsal aorta of mouse–rat chimeras, the majority of FLK1+RUNX1+ cells expressed GFP, indicating the mouse origin of these cells (Figure 5B). Later in development, percentages of mouse Lin cells, LSKs, and ST-HSCs were higher in fetal livers of mouse–rat chimeras as demonstrated by FACS analysis for Lin, CD117, Sca1, CD48, and CD150 (Figure 5C and Figure 5—figure supplement 2). The percentage of LT-HSC in fetal livers was unchanged (Figure 5C). Thus, ESC complementation causes the earlier development of donor HSCs in the dorsal aorta and increases percentages of donor-derived Lin cells, LSKs, and ST-HSCs in the fetal liver.

Figure 5 with 2 supplements see all
Mouse hematopoietic stem cells (HSCs) in mouse–rat chimeras develop earlier than rat HSCs.

(A, B) Immunostaining for RUNX1 (white) and FLK1 (red) shows that hemogenic endothelium in the dorsal aorta (DA) of mouse–rat chimeras develops mostly from embryonic stem cell (ESC)-derived mouse cells. GFP (green) was used to identify ESC-derived cells, whereas 4′,6-diamidino-2-phenylindole (DAPI, blue) was used to stain cell nuclei. Frozen sections were obtained from E11 mouse embryos and E12.5 embryos from rats and mouse–rat chimeras since these embryos are in similar developmental stages, see also Figure 5—figure supplement 1A–C. DA indicates the lumen of dorsal aorta. Yellow dashed line indicates the luminal surface of DA wall. Inserts show high magnification of hemogenic endothelial cells expressing both RUNX1 and FLK1. Scale bars are: A, 200 μm; B, 20 μm; inserts in B, 5 μm. Abbreviations: DA, dorsal aorta; Li, liver. (C) Fluorescence-activated cell sorting (FACS) analysis shows increased percentages of mouse ESC-derived Lin cells, LSKs, and short-term HSCs (ST-HSCs) in fetal livers of mouse–rat chimeras (n = 6) compared to control mouse embryos (n = 4), see also Figure 5—figure supplement 2. Fetal livers were obtained from E15.5 mouse–rat chimeras and E14 mouse embryos since these embryos are in similar developmental stages. *p < 0.05, **p < 0.01, N.S. indicates no significance, see also Source data 1.

Transplantation of ESC-derived BM cells from interspecies mouse–rat chimeras rescues lethally irradiated syngeneic mice

To test functional properties of mouse BM hematopoietic progenitor cells derived through a rat, cells were FACS-sorted for GFP from the BM of juvenile mouse–rat chimeras and transferred into the tail vein of syngeneic C57BL/6 adult mice that received the lethal dose of whole-body gamma-irradiation 3 hr prior to the BM transplant (Figure 6A). Consistent with published studies (Rowe et al., 2016; Sugimura et al., 2017; Vo and Daley, 2015), all mice without BM transplant died between 9 and 12 days after irradiation (Figure 6B). In contrast, all 20 mice transplanted with GFP+ BM cells from mouse–rat chimeras survived after lethal irradiation (Figure 6B, C). Histological assessment of femur bones confirmed the presence of GFP+ donor cells in the BM compartment of transplanted mice (Figure 6D). Blood analysis of mice harvested 8 days after irradiation showed significant decreases in white blood cells (WBCs), red blood cells (RBCs), platelets (PLT), hemoglobin (Hb) as well as numbers of granulocytes, monocytes, and lymphocytes (Figure 7A and Figure 7—figure supplements 1 and 2). Transplantation of ESC-derived BM cells from mouse–rat chimeras increased WBC and the numbers of granulocytes, monocytes, and lymphocytes in the peripheral blood at day 8 (Figure 7A and Figure 7—figure supplements 1 and 2). Contribution of ESC-derived BM cells to granulocytes, monocytes, and B cells was higher compared to erythroid and T cells (Figure 7B and Figure 7—figure supplement 3). At 5 months after BM transplantation, ESC-derived cells completely restored blood cell numbers, PLT and Hb in lethally irradiated mice (Figure 7C and Figure 7—figure supplements 1 and 2). Long-term contributions of ESC-derived BM cells to all hematopoietic cell lineages in the peripheral blood were between 49% and 96% (Figure 7C and Figure 7—figure supplement 3). Thus, transplantation of ESC-derived BM cells from mouse–rat chimeras prevented mortality and restored hematopoietic blood lineages in lethally irradiated syngeneic mice.

Transplantation of mouse embryonic stem cell (ESC)-derived bone marrow (BM) cells from interspecies mouse–rat chimeras rescues lethally irradiated syngeneic mice.

(A) Schematic diagram shows transplantation of ESC-derived bone marrow cells (BMCs) into lethally irradiated (IR) mice. ESC-derived cells were obtained from the BM of juvenile mouse–rat chimeras using fluorescence-activated cell sorting (FACS) for GFP+ cells. BM and peripheral blood were harvested 8 days and 5 months after BM transplantation. (B) Kaplan–Meier survival analysis shows a 100% mortality in irradiated mice. Survival is dramatically improved after transplantation of irradiated mice with ESC-derived BM cells obtained from mouse–rat chimeras (IR + BMC). Survival in untreated wild-type (wt) mice is shown as a control (n = 12–20 mice in each group). (C) Photograph shows irradiated C57BL/6 mice 5 months after successful BM transplantation. Untreated C57BL/6 mouse is shown as a control. Gray color of irradiated mice (arrows) is consistent with large doses of whole-body radiation treatment. (D) Hematoxylin and eosin (H&E) staining shows increased amounts of hematopoietic cells in femur bones after BM transplantation into irradiated mice (top panels). GFP+ donor cells (green) are abundant in the BM compartment of transplanted mice (bottom panels). DAPI (blue) was used for counterstaining. Scale bars are: D, 200 μm; inserts in D, 5 μm.

Figure 7 with 6 supplements see all
Transplantation of mouse embryonic stem cell (ESC)-derived bone marrow (BM) cells from interspecies mouse–rat chimeras restores hematopoietic cell lineages in the blood and BM of lethally irradiated syngeneic mice.

(A) Blood analysis shows that transplantation with ESC-derived BM cells from mouse–rat chimeras increases white blood cell (WBC) counts and red blood cell (RBC) counts in the peripheral blood of irradiated recipients. Blood samples were obtained from untreated mice (no IR), lethally irradiated mice without BM transplant (IR), and lethally irradiated mice with BM transplant (IR + BMC). BM transplantation was performed using ESC-derived BM cells obtained from juvenile mouse–rat chimeras. Fluorescence-activated cell sorting (FACS) analysis of the peripheral blood to identify granulocytes, B cells, monocytes, T cells, and erythroid cells in shown in Figure 7—figure supplement 1. Concentrations of lymphocytes, monocytes, and neutrophil in the blood were increased after BM transplantation (n = 9–15 mice in each group), **p < 0.01, N.S. indicates no significance, see also Source data 1. BM transplantation also increased concentrations of platelets, hemoglobin, basophils, and eosinophils in the peripheral blood, see Figure 7—figure supplement 2. (B, C) FACS analysis for GFP+ cells in each cell subset shows that ESC-derived BM cells from mouse–rat chimeras contribute to multiple hematopoietic cell lineages in the peripheral blood of lethally irradiated mice (n = 9–16 mice in each group), see also Figure 7—figure supplement 3. (D) FASC analysis shows that transplantation with ESC-derived BM cells from mouse–rat chimeras increases percentages of LSKs, short-term HSCs (ST-HSCs), and long-term HSCs (LT-HSCs) in the BM of irradiated mice 5 months after BM transplantation (n = 9–16 mice in each group), see also Figure 7—figure supplement 4A, B. **p < 0.01, N.S. indicates no significance, see also Source data 1. (E, F) FACS analysis for GFP+ shows that ESC-derived BM cells from mouse–rat chimeras contribute to multiple hematopoietic progenitor cells in the BM of irradiated mice (n = 9–16 mice in each group), see also Figure 7—figure supplement 5. For secondary transplantation of mouse ESC-derived BM cells into lethally irradiated syngeneic mice, see Figure 7—figure supplement 6A–E.

Transplantation of ESC-derived BM cells from interspecies mouse–rat chimeras resulted in the long-term contribution of donor cells to hematopoietic progenitor cells

Based on FACS analysis of irradiated mice at day 8, whole-body irradiation decreased the number of hematopoietic progenitor cells in the BM, including LSKs, ST-HSCs, and LT-HSCs (Figure 7D and Figure 7—figure supplement 4A, B). Transplantation of ESC-derived BM cells significantly increased LSKs but did not affect the numbers of ST-HSCs and LT-HSCs in irradiated mice (Figure 7D). Contribution of ESC-derived BM cells to Lin and LSK cell subsets was high, whereas ESC contribution to ST-HSCs and LT-HSCs at day 8 was low (Figure 7E and Figure 7—figure supplement 5). At 5 months after BM transplantation, percentages of LSKs, ST-HSCs, and LT-HSCs in the BM were increased (Figure 7D and Figure 7—figure supplement 4B). Long-term contribution of ESC-derived BM cells to LSKs, ST-HSCs, and LT-HSCs was between 92% and 95% (Figure 7F and Figure 7—figure supplement 5). Finally, we performed BM transplantation again in secondary recipients to establish the functional potential and self-renewal capacity of the chimeric HSCs (Figure 7—figure supplement 6A). The secondary BM transplantation rescued lethally irradiated mice and resulted in long-term engraftment of ESC-derived HSCs into hematopoietic cell lineages in the BM and peripheral blood (Figure 7—figure supplement 6B–E). Altogether, transplantation of ESC-derived BM cells from mouse–rat chimeras resulted in efficient, long-term contribution of donor cells to the BM and blood of lethally irradiated mice.

Discussion

Recent single-cell RNA sequencing studies identified remarkable diversity of hematopoietic cell types in the BM (Baccin et al., 2020). Generation of functional BM cells from pluripotent ESCs or iPSCs in a dish or in organoids represents a formidable challenge (Rowe et al., 2016; Vo and Daley, 2015). In the present study, we used blastocyst complementation to generate a diversity of hematopoietic cell types from mouse ESCs in rat embryos. Interspecies mouse–rat chimeras were viable and contained approximately 25% of ESC-derived mouse cells in the BM. It is possible that inactivation of genes critical for hematopoiesis in rat embryos prior to blastocyst complementation can improve the integration of mouse ESCs into the BM of mouse–rat chimeras. This approach was supported by recent studies with mouse–mouse chimeras, in which ESCs contributed to more than 90% of hematopoietic cells in mice deficient for either Kit or Flk1 (Hamanaka et al., 2018; Jansson and Larsson, 2010). While ESCs contributed to all hematopoietic cell lineages in interspecies BM, the percentage of lymphoid progenitors was lower, whereas the percentages of myeloid progenitor cells and HSCs were higher in mouse–rat chimeras compared to control mouse–mouse chimeras. Since both chimeras were produced by complementing blastocysts with mouse ESCs from the same ESC-GFP cell line, it is unlikely that these changes are dependent on donor ESCs. It is possible that the observed differences in BM cellular composition between mouse–rat and mouse–mouse chimeras are due to interactions of donor ESCs with the host embryo. Structural and functional differences between hormones, growth factors, and their receptors in rats and mice can contribute to the efficiency or timing of differentiation of mouse ESCs into hematopoietic cell lineages in BM of chimeras.

Our data demonstrate that chimeric HSCs develop more efficiently from donor mouse cells in the dorsal aorta, fetal liver, and BM, whereas rat cells are less efficient to differentiate into HSCs. Since we observed high numbers of mouse hemogenic endothelial cells in the chimeric dorsal aorta, it is likely that donor hemogenic endothelium undergoes direct transition to functional HSCs in the fetal liver, whereas endogenous (non-GFP+) hemogenic endothelium can be a source of rat HSCs. Since mouse embryos develop faster compared to rat embryos by approximately 1.5 days (Farrington-Rock et al., 2008; Marcela et al., 2012; Takahashi and Osumi, 2005; Torres et al., 2008), it is possible that mouse ESC-derived progenitor cells migrate faster into developing hematopoietic niches in the mouse–rat chimeras, leading to preferential development of HSCs from cells of mouse origin and contributing to increased numbers of mouse-derived hematopoietic progenitors in the BM of mouse–rat chimeras. These data suggest that using donor ESCs from species with less gestational time in interspecies ‘bioreactors’ can lead to larger quantities of ESC-derived hematopoietic progenitors in the chimeric BM. Our single-cell RNAseq analysis enabled us to identify potential signaling pathways and receptor–ligand interactions between hematopoietic progenitors and stromal cells in the BM. These pathways include Cxcl12-Cxcr4 signaling between hematopoietic progenitors and endothelial cells, which plays a critical role in maintenance of HSCs during BM homeostasis and promotes niche regeneration and hematopoietic reconstitution after BM transplantation (Baccin et al., 2020; Singh et al., 2020; Sugiyama et al., 2006). Other pathways identified in our studies, including Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap, have not been extensively studied in the BM microenvironment but are implicated in regulation of cell adhesion, migration, oncogenesis, fibrosis, and inflammatory responses (Kiratipaiboon et al., 2020; Sibin et al., 2019; Rock et al., 2010; Strelnikov et al., 2021; Yang et al., 2017). Notably, our data suggest that some of these signaling pathways can be targeted to modulate the development and expansion of donor ESC-derived hematopoietic progenitor cells in the BM of interspecies chimeras.

Despite mosaicism in interspecies BM, mouse ESC-derived cells from multiple hematopoietic cell lineages were highly differentiated and indistinguishable from the normal mouse BM cells based on gene expression signatures and cell surface proteins. Consistent with functional competency of ESC-derived BM, transplantation of BM cells into lethally irradiated syngeneic mice prevented mortality and resulted in long-term contribution of ESC-derived cells to all hematopoietic cell lineages in the BM and peripheral blood. One of the limitations of our studies is that the functional potential of chimeric HSCs was established from whole BM transplants and not from transplantation of purified HSCs. While these experiments are technically challenging, transplantation of FACS-sorted donor HSCs into lethally irradiated mice will be needed in our future studies to investigate whether chimeric HSCs are fully functional to restore all hematopoietic cell lineages after irradiation. Our results are consistent with recent studies demonstrating the ability of mouse ESCs to generate functional pancreatic, endothelial, and kidney cells in interspecies mouse–rat chimeras (Goto et al., 2019; Wang et al., 2021; Yamaguchi et al., 2017). Interestingly, long-term contribution of donor BM cells to ST-HSCs and LT-HSCs of irradiated mice was high, supporting the ability of donor HSCs to self-renew. In contrast, the short-term contribution of donor BM cells to ST-HSCs and LT-HSCs of irradiated mice was low. Low contribution of donor BM to HSCs at day 8 is not surprising considering an acute hematopoietic deficiency in lethally irradiated mice. It is possible that most donor-derived HSCs undergo rapid differentiation into other hematopoietic cell types to compensate for the loss of injured hematopoietic cells after irradiation.

Generation of intraspecies chimeras through blastocyst complementation creates an interesting opportunity to use patient-derived iPSCs to produce tissues or even organs in large animals, for example, pigs or sheep, which can serve as ‘biological reactors’. However, at this stage of technological advances it is impossible to restrict the integration of ESC/iPSC-derived cells into selected organs or cell types. Off-target integration of ESCs and iPSCs into the brain, testes, and sensory organs raises important ethical concerns for the use of human–animal chimeras in regenerative medicine (Masaki and Nakauchi, 2017; Wu et al., 2016). To improve the selectivity of ESC/iPSC integration into chimeric tissues, various genetic modifications can be introduced into the host embryos to advance the technology. Harvest of tissues from chimeric embryos instead of adult chimeras can alleviate some of the ethical concerns, suggesting a possibility of using chimeric embryos as a potential source of patient-specific hematopoietic progenitor cells.

In summary, blastocyst complementation of rat embryos with mouse ESCs was used to simultaneously generate multiple hematopoietic and stromal cell lineages in the BM. ESC-derived cells in mouse–rat chimeras were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells rescued lethally irradiated syngeneic mice and resulted in long-term contribution of donor cells to hematopoietic cell lineages. Thus, the interspecies chimeras could be considered for in vivo differentiation of patient-derived iPSCs into hematopoietic cell lineages for future cell therapies.

Materials and methods

Mice, rats, and generation of mouse–rat and mouse–mouse chimeras through blastocyst complementation

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C57BL/6 mice were purchased from Jackson Lab. Interspecies mouse–rat chimeras were generated using blastocyst complementation as described (Li et al., 2021; Wang et al., 2021). Briefly, blastocysts from SD rats were obtained at embryonic day 4.5 (E4.5), injected with 15 GFP-labeled mouse ESC cells (ESC-GFP, C57BL/6 background) (Sun et al., 2021; Wen et al., 2021) and transferred into pseudopregnant SD rat females. Mouse–mouse chimeras were generated by complementing CD1 blastocysts with 15 mouse ESC-GFP cells. For FACS analysis and BM transplantation, BM cells were collected from chimeric pups that were harvested between postnatal day 4 (P4) and P10. For single-cell RNA sequencing, BM cells were prepared from P10 and P5 mice, rats, and chimeras. To perform BM transplantation, BM cells from two tibias and two fibulas of mouse–rat chimeras were collected and FACS-sorted for ESC-derived (GFP+) cells. 500,000 FACS-sorted GFP+ BM cells were intravenously (i.v.) injected into lethally irradiated C57BL/6 male mice (6–8 weeks of age) via the tail vein. Three hours before BM transplantation, whole-body irradiation was performed using 11.75 Gy. Mice were harvested after 8 days or 5 months after BM transplantation. For the second BM transplantation, GFP+ BM cells were FACS-sorted from irradiated mice 5 months after the first BM transplantation and then i.v. injected into new irradiated recipients. Tissue dissection, processing, and preparation of single-cell suspensions were carried out as described (Bolte et al., 2011; Kalin et al., 2008; Kalinichenko et al., 2003; Kim et al., 2005; Wang et al., 2003). Blood analysis was performed in animal facility of Cincinnati Children’s Hospital Research Foundation.

Single-cell RNAseq analysis of ESC-derived BM cells

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Prior to scRNAseq (10× Chromium platform), BM cells were pooled from three P10 mouse–rat chimeras and three P10 mouse–mouse (control) chimeras and then FACS-sorted for GFP and the lineage (Lin) marker. Since the numbers of HSCs and other hematopoietic progenitors in BM are significantly low compared to numbers of differentiated hematopoietic cells, the cell mixtures were enriched for BM progenitor cell populations by combining 90% of FACS-sorted GFP+Lin cells and 10% of GFP+Lin+ cells in each experimental group. This enrichment enabled us to obtain enough progenitor cells for UMAP clustering analysis. In separate scRNAseq experiments, all BM cells (including hematopoietic, vascular, and stromal cells) were prepared from P5 mice, rats, and mouse–rat chimeras using enzymatic digestion and cell purification as described (Baccin et al., 2020). BM cells from five animals were pooled together prior to single-cell RNAseq. All raw data and the processed count matrix of BM datasets were uploaded to the GEO database (accession number GSE184940). Read alignments, quality controls, and false discovery rates were described previously (Guo et al., 2019; Ren et al., 2019; Wang et al., 2022). Identification of cell clusters and quantification of cluster-specific gene expression in BM scRNAseq datasets were performed as described (Baccin et al., 2020; Wang et al., 2021; Wen et al., 2021). To assess the transcriptomic similarity of ESC-derived and endogenous BM cells, the scRNAseq datasets were normalized with SCTransform and then integrated utilizing the canonical correlation analysis. In the integrated scRNAseq datasets, the SelectIntegrationFeatures in Seurat package (version 4.0.0 in R 4.0 statistical environment) was used to identify anchors for integration. The RunPCA function was used for principal component analysis (PCA) of scRNAseq datasets, and the PCElbowPlot function was used to calculate the standard deviations of the principal components (PCs). PCs with standard deviation >3.5 were chosen as input parameters for nonlinear UMAP clustering analysis. Next, the FindNeighbors function was used to compute the k.param nearest neighbors, and BM cell clusters were identified by a shared nearest neighbor modularity optimization clustering algorithm implemented in the FindClusters function with resolution set at 0.4 (Guo et al., 2019; Wang et al., 2021; Wen et al., 2021).

Analysis of potential receptor–ligand interactions using single-cell RNAseq datasets

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The R package NicheNet was used to analyze the information about expression of cognate ligands and receptors to identify intercellular communication patterns between hematopoietic progenitors and stromal cells as described (Browaeys et al., 2020). EMP and GMP cells were chosen as potential sources of receptors, whereas BM stromal cell types were chosen as potential sources of ligands. The background expression of genes was specified with default approach used in the NicheNet pipeline, and expressed genes were identified based on >10% detection in specific clusters. To identify ligand–receptor interactions between EMPs/GMPs and stromal cells, we selected the top 20 ligands predicted to drive hematopoietic cell differentiation based on the Pearson correlation coefficient between the ligand–receptor regulatory potential score of each ligand and the target indicator vector. Using the NicheNet pipeline, the Circos plots were generated to show common ligand–receptor interactions between EMPs/GMPs and stromal cells in the BM.

FACS analysis

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FACS analysis was performed using cells obtained from the BM and blood. Antibodies for FACS analysis are listed in Supplementary file 3. Immunostaining of cell suspensions were performed as described (Bolte et al., 2017; Xia et al., 2015). Identification of hematopoietic cell types based on multiple cell surface markers is described in Bolte et al., 2020b; Pradhan et al., 2019; Ren et al., 2013; Ren et al., 2010; Sun et al., 2017. To identify ESC-derived HSCs, we used GFP fluorescence and mouse-specific antibodies recognizing multiple cell surface antigens. First, ESC-derived GFP+ BM cells were subdivided into Lin+ and Lin cell subsets. Second, we used Sca1 and CD117 (c-KIT) antibodies to identify LinSca1+c-KIT+ cells (LSKs). Third, CD150 and CD48 antibodies were used to identify ST-HSCs and LT-HSCs among LSKs. Stained cells were analyzed using a five-laser FACSAria II (BD Biosciences) (Cai et al., 2016; Sun et al., 2021).

Histology and immunostaining

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Frozen or paraffin-embedded sections of tissue samples were stained with hematoxylin and eosin (H&E) for histological evaluation (Kalinichenko et al., 2002) or to visualize GFP (Ustiyan et al., 2018; Ustiyan et al., 2016). Frozen sections from embryos were used for immunofluorescent staining as described (Black et al., 2018; Ustiyan et al., 2012; Wang et al., 2010). Primary antibodies for immunostaining are listed in Supplementary file 3. Secondary antibodies were conjugated with Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647 (Invitrogen and Jackson ImmunoResearch Laboratory) to visualize specific staining as described (Bolte et al., 2012; Hoggatt et al., 2013; Milewski et al., 2017a). DAPI (Vector Laboratory) was used to counterstain cell nuclei (Milewski et al., 2017b). Histological and immunofluorescent images were obtained using a Zeiss Axioplan2 microscope (Carl Zeiss Microimaging) as described (Bolte et al., 2015; Kalin et al., 2008; Pradhan et al., 2016).

Statistical analysis

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Statistical significance was determined using nonparametric Mann–Whitney U-test, one-way analysis of variance, and Student’s t-test. Multiple means were compared using one-way analysis of variance with the post hoc Tukey test. p ≤ 0.05 was considered statistically significant. Data were presented as mean ± standard error of mean (SEM).

Data availability

Bone marrow single-cell RNA sequencing data have been deposited in GEO under accession number GSE184940.

The following data sets were generated
    1. Wen B
    2. Wang G
    3. Kalinichenko VV
    (2021) NCBI Gene Expression Omnibus
    ID GSE184940. The integrated single cell RNAseq analysis of bone marrow cells produced by mouse-mouse intraspecies blastocyst complementation and mouse-rat interspecies blastocyst complementation.

References

Decision letter

  1. Jalees Rehman
    Reviewing Editor; University of Illinois at Chicago, United States
  2. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  3. Jalees Rehman
    Reviewer; University of Illinois at Chicago, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for sending your article entitled "in vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras" for peer review at eLife. Your article is being evaluated by 3 peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Didier Stainier as the Senior Editor.

The reviewers were impressed by the chimera assay because it has the potential to uncover significant mechanisms of hematopoiesis and how specific niche interactions instruct hematopoiesis. However, the reviewers also felt that in its present form, the manuscript established the chimera model without showing its utility for gaining specific new mechanistic insights.

To leverage your blastocyst chimera model and derive important new insights, there would be the need for new analyses of the scRNA-seq data as well as new experimental studies that result in mechanistic insights. Your action plan for addressing these comments would help us reach a definitive decision.

Reviewer #1 (Recommendations for the authors):

Wen et al., establish the blastocyst complementation assay as a bioreactor; that helps direct the differentiation of mouse ESCs into the complete hematopoietic lineages. The elegance of the approach lies in the use of GFP+ mouse ESCs that are implanted into a rat blastocyst, thus allowing for the tracking and phenotyping of the mouse derived GFP+ hematopoietic cells in the post-natal rat. Single cell RNA-sequencing, flow cytometry and serial transplantation convincingly demonstrate that mouse ESCs differentiate into true HSCs as well as the more mature hematopoietic lineages. This further shows that the rat blastocyst can serve as a bioreactor which provides all the necessary cues to direct mouse ESC differentiation. The blastocyst complementation assay could therefore serve as a starting point to unravel the niche cues required for ESC differentiation into HSCs and hematopoietic progenitors as well generation of ex vivo bioreactors to generate and expand fully functional HSCs.

Key strengths:

1. The use of GFP+ ESCs is an elegant approach to track the fate of mouse ESCs.

2.The single cell RNA-seq analysis and flow cytometry data show the full spectrum of differentiation of mESCs.

3. Transplantation of mESC derived GFP+ cells nicely demonstrates that the generated HSCs are fully functional.

4. The described blastocyst complementation assay could be a useful platform for dissecting mechanisms of niche cues that direct ESC differentiation into hematopoietic lineages.

Key weaknesses:

1. A major feature of a "bioreactor" is the understanding and characterization of its key cellular components or molecules that drive its function. While the blastocyst complementation assay is very successful in generating fully functional HSCs and HSC progeny by the time cells are analyzed post-natally at P10, it is unclear how the blastocyst provides this information and, specifically, which cells or molecules in the blastocyst are essential for ESC differentiation into hematopoietic lineages.

2. The bioinformatic analysis of single cell RNA-seq data from GFP+ cells primarily uses general clustering and correlation analyses to assess similarity of mouse-rat chimera vs control. However, these analyses are performed at P10 and do not provide any clear insights or even suggestions as to how the blastocyst environment might have guided mouse ESCs to differentiation into hematopoietic lineages during the earlier developmental timepoints.

There are some key suggestions that would really help increase the impact of the work:

Can the single cell analysis be performed during one or more jey developmental timepoints after blastocyst complementation so that one could perhaps obtain insights into how the blastocyst begins to program ESCs during development?

Separating GFP+ and GFP- cells in the developing embryo at a timepoint when hematopoiesis occurs from a defined hematopoietic tissue such as the fetal liver could provide important insights into interactions between mESCs and the blastocyst role as a niche, as well as which niche components form from the GFP+ implanted ESCs.

Furthermore, a more in-depth analysis of predicted ligand-receptor interactions (using standard databases of ligand-receptor pairs) with inclusion of potential niche cells (stromal cells, endothelial cells, etc) that guide hematopoiesis in the scRNa-seq because they likely provide ligands for hematopoietic cells, and the inference or prediciton of transcription factor activities in the various cells during embryogenesis would also help inform how the blastocyst is driving the ESC differentiation towards hematopoietic cells.

Such experiments would leverage the elegance of the GFP+ mESC implantation and lineage tracing to identify specific cell types and pathways that one could modulate in order to understand novel mechanisms by which the "bioreactor" functions.

Reviewer #2 (Recommendations for the authors):

In the current study the authors utilize a well-developed system of interspecies chimaera to show conclusively that murine embryonic stem cells implanted in a rat blastocyst to produce viable mouse-rat chimaeras. This finding is strengthened by two observations: (1). The single-cell transcriptomic profile of the mouse-rat chimaeras closely matches that of the control mouse-mouse chimaeras for both the HSCs/progenitors as well as individual lineages. (2). The functional potential of mouse-rat chimaeras is tested by transplantation assays that show the chimaera derived cells to be capable of multi-lineage reconstitution. These two aspects of the study are both carefully conducted and supported the conclusion that the chimeric system, in the bone marrow niche, faithfully replicates the physiological and functional development of hematopoiesis.

While there are a few technical details that need to be addressed, the main critique of the study is the fact that characterization of hematopoiesis is limited to the bone marrow of newborn mouse-rat chimaeras. As the authors themselves admit, the blastocyst complementation system has already been shown to produce bone marrow hematopoietic cells in mouse-mouse chimaeras with added complexity of transgenic mutants (Hamanaka S et al., 2018), thus the observation that their mouse-rat chimaeras produce functional bone marrow HSCs is not inherently novel and primarily relies on the careful characterization of single cell transcriptional profiles to indicate that the mouse ESCs have produced transcriptionally authentic HSCs.

What the study needs in order to stand out and make ideal use of the mouse-rat chimeric system is a developmental perspective of how the murine ESCs are converted or incorporated into the embryo in order to produce definitive murine HSCs from the hemogenic endothelium of dorsal aorta. Or alternatively a better characterization, functional or transcriptional of the fetal GFP+ murine HSCs during maturation in the fetal liver.

It would be very enlightening to determine if the murine tissue must transition directly from the hemogenic endothelium to functional HSCs or if it can emerge denovo from non-GFP+ hemogenic endothelium. Since the cells are GFP-labeled and clearly identifiable from the rat tissue, this analysis can be performed by flow cytometry, single-cell RNAseq or even imaging and microscopy. It is not known when and how the chimeric system produces definitive HSCs in the embryos and the system that investigators have utilized to study the adult bone marrow is capable of answering this important question and thus producing a truly novel study.

The authors have done a commendable job characterizing the bone marrow contribution of the blastocyst complementation system in their mouse-rat chimaeras. Their results clearly show the amount and the expression profiles GFP+ murine HSCs and progenitors. However, on its own this characterization is not enough to merit publication as it is not mechanistically novel and makes use of previously established systems and only incrementally advances the field by using the chimeric system in an inter-species model.

The single cell RNAseq is informative and drives home the point that the murine ESCs produce the HSCs that are transcriptionally similar to mouse-mouse chimaeras, but the fact that the cells are capable of reconstitution in a transplant setting is already sufficient to establish the functional potential of the mouse HSCs in a rat chimeric bone marrow. Thus, a mechanistic and/or developmental approach is required to determine when and how the mouse ESCs produce the definitive HSCs in the chimeric fetus. This can already be performed with the system that the authors have in hand and only requires timed matings and chimeric embryo extraction and characterization of the fetal GFP+ cells either in the AGM region of the dorsal aorta during HSC emergence or the fetal liver during HSC expansion.

It would be very interesting to determine if the mouse HSCs emerge at the same rate as the rat ones or if there is a different selection during emergence and expansion in the fetal liver that produces the high frequency of GFP+ LT-HSCs seen in the bone marrow (Figure 1H). This integration and incorporation of the mouse cells in the chimeric system during embryo development would truly set the story apart and provide novel findings essential to our understanding of developmental hematopoiesis and chimeric model systems in general.

Specific comments:

In Fig,1, for both the legend and the text of the manuscript, the use of the term "control" and "mouse" is used interchangeably, but it is difficult to understand what it refers to. The comparison of a WT mouse to a GFP+ mouse-rat chimaera is not ideal as a control in this case because in a mouse presumably all the HSCs and progenitors are detectable, while in a mouse-rat chimaera only a subset of the cells are GFP+HSCs. This is very evident in Figure 1F where the "mouse" seems to have no GFP+ BM contribution in the BM while the chimaera has on average 25% contribution of GFP+ cells in the BM. It is not obvious why this is the case? A better control would be the assessment and comparison of the rat HSCs and progenitors in the mouse-rat BM of the chimeric mice.

In Figure 3 and Figure 4 how does the expression of the endogenous rat HSCs compare to that of the mouse HSCs in the chimaera. Presumably these two similar cell types from different species share a common bone marrow niche and thus are expected to be similar unless the presence of the mouse GFP+ HSCs and progenitors affects the endogenous rat cells.

Whole bone marrow transplants (with 500,000 GFP+ cells) are not the ideal means to establish cell autonomous functionality of the HSCs. For this purpose, phenotypic GFP+ HSCs, as shown in Figure 1E, should be sorted and transplanted into irradiated donors. Ideally, the transplant should be performed once again in secondary recipients to fully establish the functional potential and self-renewal capacity of the chimeric HSCs.

Figure 6A is not necessary since it is entirely subjective and has no values listed in any of the chosen gates. It should be moved to the supplement and Figure 5 should then be combined with Figure 6.

Figure 7A is not informative and should be removed from the manuscript. Instead, the authors should perform longitudinal sections of the bone (femur) and stain for H and E to show the effects of IR on the bone marrow microenvironment in the listed timepoints and treatments.

Reviewer #3 (Recommendations for the authors):

Rat-mouse interspecies chimeras have been used for many years and are useful to study the impact of various gene deficiencies in reconstituting different organs or tissues by mixing mutated and wild type cells. Here the authors have focused on the bone marrow and more particularly on the hematopoietic compartment.

The paper by Weng et al., aims at investigating the formation of the bone marrow with a focus on the hematopoietic compartment. They are using rat-mouse interspecies chimeras where mouse embryonic stem cells GFP-tagged are injected into rat blastocysts that subsequently develop into viable chimeric animals after reimplantation into pseudo-pregnant females. The authors sort the bone marrow cells from the chimeras on the basis of GFP and analyzed the hematopoietic cell populations with the standard flow cytometry approaches and reconstitution analysis in irradiated recipients. They conclude that a mouse-derived complete and functional hematopoietic hierarchy is present in the chimeric animals with an increase of short-term and long-term mouse hematopoietic stem cells compared to non-injected mice.

They analyzed the hematopoietic cell populations using single cell RNA sequencing and found weak or no difference compared to non-chimeric animals and they probed the reconstitution potential of the hematopoietic stem cell compartment. This is an interesting study that is essentially descriptive regarding the system of chimerism. The authors have however biased the analysis by focusing on the hematopoietic compartment and not on the entire bone marrow as they claim. Of note, the significant increase in the hematopoietic stem cell compartment observed in the chimeric context has not been analyzed. This is a key point of the study since it may help isolating factors to amplify hematopoietic stem cells. Thus despite an interesting design, this study falls short in finding key mechanisms of hematopoietic stem cell homeostasis and self-renewal.

Strengths

Exploring alternative approaches for the production of transplantable hematopoietic stem cells for therapeutic purposes.

Mastering interspecies rat-mouse chimeras to study the functioning of species-specific hematopoietic systems in a mixed bone marrow environment.

Weaknesses

Short, mostly descriptive study with few insights into how to improve hematopoietic stem cell manipulation and amplification.

Bias in the analysis by using only the hematopoietic fraction of the GFP+ population. The authors claim they are studying the bone marrow but they are in fact analyzing the hematopoietic compartment. The stromal cell compartment is totally lacking.

The single cell approach is biased since authors are artificially mixing different percentages of lin+ and lin- cells. The most interesting part of the work i.e., the amplification of short-term and long-term hematopoietic stem cells in chimeras should not be approached this way.

The long term reconstitution lacks secondary transplantation to show that hematopoietic stem cells are endowed with long-term reconstitution potential.

This is an interesting study that might give clues into how create and/or amplify hematopoietic stem cells.

Please focus on the entire bone marrow rather than on the hematopoietic cell compartment.

Analyze the transcriptome without introducing bias. The idea is to understand how the hematopoietic stem cell compartment is expanded in chimeras, not to show that all the hematopoietic cell lineages are present.

Analyze the stromal cell compartment that is complementary to the hematopoietic stem cell compartment.

Perform secondary transplantation to show that secondary reconstitution can take place.

Expand your analysis to cytokines and growth factors.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "in vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras" for further consideration by eLife. Your revised article has been evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1. The concern raised by Reviewer 2 about the functional potential of the GFP+ mouse BM HSCs from the chimeric mice.

2. Addressing the comments of Reviewer 3 about the presentation of specific figure panels to illustrate the key points of the study in a cohesive manner and the need for sufficient rationale + context.

It is likely that the questions can be addressed with existing data which needs to be prioritized (main figures versus supplement), presented in a cohesive manner, elaborated (experimental details) on and discussed more extensively (whenever there are limitations which affect the conclusions).

Reviewer #1 (Recommendations for the authors):

The authors have substantially expanded the manuscript through highly relevant new studies in response to the reviewer comments and have thereby increased the robustness and significance of their findings.

The significant improvements include the addition of more time points, the analysis of putative ligand receptor interactions and the secondary bone marrow transplantation studies.

The scRNA-seq data have been uploaded to GEO and are accessible to the public.

Reviewer #2 (Recommendations for the authors):

Overall manuscript is much improved, has broader appeal and now provides very intriguing findings on the developmental perspective of when HSCs emerge in a chimeric embryo as a potential timing regulation of LT-HSCs expansion in the fetal liver.

The authors were mostly responsive to the critiques and have sufficiently improved the novelty of the manuscript by performing experiments in the developing embryo.

However, there is one remaining critical concern regarding the functional potential of the GFP+ mouse BM HSCs from the chimaera. The authors say that they attempted to transplant sorted GFP+ stem cells but only about 198 LT-HSCs and ST-HSC were purified on average per chimaera BM. Furthermore, they say that these cells didn't provide enough hematopoietic support for a viable recipient mouse. This is not a valid response and raises a very big question about the cell intrinsic functional potential of the GFP+ mouse BM HSCs. Considering that 500,000 total cells provide reconstitution even long-term secondary transplant reconstitution, and with Figure 1H showing that there are chimaeras with as many as 40% GFP+ ST-HSCs and LT-HSCs, the ability to conduct a transplant with purified HSCs should be possible.

The authors did not have to test the average of GFP+ HSCs per chimaera, they simply had to show that some of the GFP+ cells were capable of cell autonomous long-term reconstitution. This brings to question the absolute number of mouse GFP+ HSCs in the chimaera bone marrow. Which should be determined from their flow cytometric analysis. The authors have to explain how they purified the ST- and LT-HSCs from the bone marrow and why 198 could not provide them with a viable recipient mouse when work from the Weissman and Morrison labs have shown that as few as 10 LT-HSCs (SLAM LSKs) can provide functional hematopoiesis in a transplant setting. Did the authors support the recipient mice with lineage+ splenocytes? What was the dose of irradiation administered? There has to be a better description of this experiment in the methods, in fact the methods as listed in the revision are very light on details of transplantation and lineage analysis.

So while the functional potential of chimeric stem cells has been established from the bulk bone marrow transplants, the lack of purified GFP+ mouse HSCs transplants limits the conclusions that the authors make with regard to calling these mouse HSCs functional in their experimental setting.

Reviewer #3 (Recommendations for the authors):

This reviewer thanks the authors for the revised version of their manuscript that shows significant improvement.

On the basis of this revised version, you will find enclosed my main comments for manuscript improvement. In general, I have the feeling that some of the figures are redundant or that the authors have trouble making a choice on what they want to show.

Figure 1: The authors examine the percentages of the ST-HSC and LT-HSC in WT mice vs chimeric animals. Ideally, a mouse/mouse chimeric control should also be included. In the Sca-Kit analysis (Fig1E); the authors mentioned a significant increase in the Sca-Kit compartment in chimeras vs WT animals. This is substantiated in Fig1G, although two groups of animals with distinct (high and low) percentages of Sca-Kit are visible but not substantiated in Figure 1E where the authors show close percentages between the two conditions. The same remark applies in the opposite direction for the percentages of LT vs ST HSC. I would recommend more carefully choosing the panels to illustrate their conclusions. The rationale of analyzing the animals at P10 is rather uncommon in hematology unless you want to analyze the forming bone marrow. P30 would have been more classical.

Figure 2: The design of this experiment is still puzzling for me and is not clearly indicated either in the text or in the legend. The authors have sorted GFP+ cells from the BM. Why is it necessary to enrich in committed hematopoietic progenitors since the BM already contains high numbers of these cells? Please clarify. The rationale of mixing mouse/mouse and mouse/rat GFP+ cells is not clear since the authors do not identify cells originating from one combination vs the other. At this point, instead of performing a scRNA-seq, a GFP+ FACS sorting followed by a multilineage hematopoietic analysis by flow cytometry i.e.myeloid, lymphoid, erythroid cells would have been more simple and direct. Could you please clarify the design of the experiment?

More generally the authors strongly emphasize on the transcriptome analysis. Several main figures are redundant and could be switched to supplementary ones e.g. Figure 3 and Figure 4 since these latter does not bring any specific insights into species-specific mechanisms.

Fig6 should be revised. The sections of the embryonic aorta are not informative regarding the endothelium and the hematopoietic cell production. Please provide images showing low magnification of the aorta and the surrounding structures and high magnification of the hematopoietic clusters.

Fig8: Multilineage analysis of the blood is interesting but the most informative piece of data comes from the BM analysis. Showing results at 8 days post-irradiation is not very informative since the animals are in aplasia.

https://doi.org/10.7554/eLife.74018.sa1

Author response

Reviewer #1 (Recommendations for the authors):

Wen et al., establish the blastocyst complementation assay as a bioreactor; that helps direct the differentiation of mouse ESCs into the complete hematopoietic lineages. The elegance of the approach lies in the use of GFP+ mouse ESCs that are implanted into a rat blastocyst, thus allowing for the tracking and phenotyping of the mouse derived GFP+ hematopoietic cells in the post-natal rat. Single cell RNA-sequencing, flow cytometry and serial transplantation convincingly demonstrate that mouse ESCs differentiate into true HSCs as well as the more mature hematopoietic lineages. This further shows that the rat blastocyst can serve as a bioreactor which provides all the necessary cues to direct mouse ESC differentiation. The blastocyst complementation assay could therefore serve as a starting point to unravel the niche cues required for ESC differentiation into HSCs and hematopoietic progenitors as well generation of ex vivo bioreactors to generate and expand fully functional HSCs.

We would like to thank the Reviewer for positive assessment of our manuscript and for recognizing future potential of our work.

Key strengths:

1. The use of GFP+ ESCs is an elegant approach to track the fate of mouse ESCs.

2.The single cell RNA-seq analysis and flow cytometry data show the full spectrum of differentiation of mESCs.

3. Transplantation of mESC derived GFP+ cells nicely demonstrates that the generated HSCs are fully functional.

4. The described blastocyst complementation assay could be a useful platform for dissecting mechanisms of niche cues that direct ESC differentiation into hematopoietic lineages.

We would like to thank the Reviewer for summarizing key strengths of our manuscript.

Key weaknesses:

1. A major feature of a "bioreactor" is the understanding and characterization of its key cellular components or molecules that drive its function. While the blastocyst complementation assay is very successful in generating fully functional HSCs and HSC progeny by the time cells are analyzed post-natally at P10, it is unclear how the blastocyst provides this information and, specifically, which cells or molecules in the blastocyst are essential for ESC differentiation into hematopoietic lineages.

We agree. We provided additional experimental data in new Figures 4, 5, 6, and 7D, and new Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2, Figure 6 —figure supplements 1A-C and 2, Figure 8 —figure supplement 6A-E. that characterize both hematopoietic and stromal cellular components in the chimeric bone marrow and analyze receptor-ligand interactions between hematopoietic and stromal cells in the chimeras. We also characterized the development of donor hematopoietic progenitors during early neonatal and embryonic time points in the bone marrow, dorsal aorta and liver of the mouse-rat chimeras. Specific new findings are summarized below in recommendations for the authors.

2. The bioinformatic analysis of single cell RNA-seq data from GFP+ cells primarily uses general clustering and correlation analyses to assess similarity of mouse-rat chimera vs control. However, these analyses are performed at P10 and do not provide any clear insights or even suggestions as to how the blastocyst environment might have guided mouse ESCs to differentiation into hematopoietic lineages during the earlier developmental timepoints.

We agree. We provided additional bioinformatic analysis of new single-cell RNA sequencing of P5 bone marrow in the mouse, rat and mouse-rat chimera which includes both hematopoietic and stromal cells of mouse (GFP+) and rat origin (GFP-) (new Figure 4 and Figure 4 —figure supplement 1A-D). We also expanded the bioinformatic analysis to analysis of receptor-ligand interactions between hematopoietic and stromal cells in the rat and mouse compartment of the chimeras (Figure 5 and Figure 5 —figure supplements 1 and 2). Finally, we characterized the contribution of mouse ESCs to HSCs during embryonic development of mouse-rat chimeras by providing new immunostaining of dorsal aorta (new Figure 6A) and FACS analysis of the fetal liver (new Figure 6B and Figure 6 —figure supplements 1A-C and 2).

There are some key suggestions that would really help increase the impact of the work:

Can the single cell analysis be performed during one or more jey developmental timepoints after blastocyst complementation so that one could perhaps obtain insights into how the blastocyst begins to program ESCs during development?

We followed up on this excellent suggestion. During these experiments, we determined that the number of cells from the bone marrow compartment of embryos is very limited and insufficient to perform single-cell RNA seq. Therefore, we performed an additional single-cell RNA seq using the early neonatal time-point (P5). Analysis of receptor-ligand interactions between hematopoietic and stromal cells in the rat and mouse compartment of the chimeras was performed and provided in (Figure 5 and Figure 5 —figure supplements 1 and 2). Furthermore, we used developmental time-points E12.5 and E15.5 from mouse-rat chimeras to characterize the contribution of mouse ESCs to HSCs by immunostaining (new Figure 6) and FACS analysis (new Figure 6B and Figure 6 —figure supplements 1A-C and 2).

Overall, these new data demonstrate that chimeric HSCs develop earlier and more efficiently from donor mouse ESCs compared to endogenous rat ESCs. Since mouse embryos develop faster compared to rat embryos by approximately 1.5 days (new references: Farrington-Rock et al., 2008; Marcela et al., 2012; Takahashi and Osumi, 2005; Torres et al., 2008; and new Figure 6 —figure supplement 1A-C), it is possible that mouse ESC-derived progenitor cells migrate faster into developing hematopoietic niches in the mouse-rat chimeras, leading to preferential development of HSCs from cells of mouse origin and contributing to increased numbers of mouse-derived hematopoietic progenitors in the bone marrow of mouse-rat chimeras. We have included this possibility in the Discussion section (pages 14-15).

Separating GFP+ and GFP- cells in the developing embryo at a timepoint when hematopoiesis occurs from a defined hematopoietic tissue such as the fetal liver could provide important insights into interactions between mESCs and the blastocyst role as a niche, as well as which niche components form from the GFP+ implanted ESCs.

We agree. As mentioned above, we analyzed separately GFP+ and GFP- hematopoietic cells and their interactions with stromal cells in P5 bone marrow (new Figures 4 and 5, and new Figure 4 —figure supplement 1A-D and Figure 5 —figure supplements 1 and 2). The bone marrow is more relevant to our study than embryonic liver tissue because of fundamental differences in hematopoietic niche components in the bone marrow compared to the liver, which contains hepatocytes, hepatoblasts, bile duct epithelial cells, stellate cells and other liver-specific cell types that are absent in cellular niches of the bone marrow.

Furthermore, a more in-depth analysis of predicted ligand-receptor interactions (using standard databases of ligand-receptor pairs) with inclusion of potential niche cells (stromal cells, endothelial cells, etc) that guide hematopoiesis in the scRNa-seq because they likely provide ligands for hematopoietic cells, and the inference or prediciton of transcription factor activities in the various cells during embryogenesis would also help inform how the blastocyst is driving the ESC differentiation towards hematopoietic cells.

We agree. As requested by the Reviewer, we provided in-depth analysis of predicted receptor-ligand interactions between hematopoietic and niche cells (including stromal cells, endothelial cells, chondrocytes, etc.) in the rat and mouse compartment of the chimeric bone marrow (new Figure 5 —figure supplement 1). We also compared gene expression signatures and the expression of key ligands and their receptors between mouse and rat cells in the chimeric bone marrow (new Figure 5 —figure supplement 2). These new data were incorporated into the revised manuscript (page 10).

Such experiments would leverage the elegance of the GFP+ mESC implantation and lineage tracing to identify specific cell types and pathways that one could modulate in order to understand novel mechanisms by which the "bioreactor" functions.

We agree. Using the GFP+ mESC implantation into rat blastocysts followed by the lineage tracing of mESC-derived cells, we identified several specific signaling pathways between hematopoietic progenitors and stromal cells which can be involved in the function of the “bioreactor”. Focusing on interaction between erythroid-myeloid progenitor cells (EMPs) and stromal cells in the bone marrow, we identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6 and Comp-Cd47 receptor-ligand pairs as major predicted interactions between EMPs and endothelial cells, fibroblasts and chondrocytes (new Figure 5). The major signaling pathways between granulocyte-myeloid progenitor cells (GMPs) and stromal cells were Col1a1-Cd44, App-Il18rap and Cxcl12-Cxcr4 (new Figure 5 —figure supplement 1). We included these data in the Results section (page 10). We also provided new references (Baccin et al., 2020; Singh et al., 2020; Sugiyama et al., 2006; Kiratipaiboon et al., 2020; Mk et al., 2019; Rock et al., 2010; Strelnikov et al., 2021; Yang et al., 2017), which support the potential importance of these pathways in cell signaling between hematopoietic and stromal cells. Finally, we modified the Discussion section to suggest that these pathways can be targeted to modulate the development of donor ESC-derived hematopoietic progenitor cells in the “bioreactor” (page 15).

Reviewer #2 (Recommendations for the authors):

In the current study the authors utilize a well-developed system of interspecies chimaera to show conclusively that murine embryonic stem cells implanted in a rat blastocyst to produce viable mouse-rat chimaeras. This finding is strengthened by two observations: (1). The single-cell transcriptomic profile of the mouse-rat chimaeras closely matches that of the control mouse-mouse chimaeras for both the HSCs/progenitors as well as individual lineages. (2). The functional potential of mouse-rat chimaeras is tested by transplantation assays that show the chimaera derived cells to be capable of multi-lineage reconstitution. These two aspects of the study are both carefully conducted and supported the conclusion that the chimeric system, in the bone marrow niche, faithfully replicates the physiological and functional development of hematopoiesis.

We would like to thank the Reviewer for positive assessment of our manuscript and for the comment that our interspecies chimeric system replicates the physiological and functional development of hematopoiesis.

While there are a few technical details that need to be addressed, the main critique of the study is the fact that characterization of hematopoiesis is limited to the bone marrow of newborn mouse-rat chimaeras. As the authors themselves admit, the blastocyst complementation system has already been shown to produce bone marrow hematopoietic cells in mouse-mouse chimaeras with added complexity of transgenic mutants (Hamanaka S et al., 2018), thus the observation that their mouse-rat chimaeras produce functional bone marrow HSCs is not inherently novel and primarily relies on the careful characterization of single cell transcriptional profiles to indicate that the mouse ESCs have produced transcriptionally authentic HSCs.

To increase the novelty of our study, we performed additional single-cell RNA sequencing experiments to compare cells in the bone marrow of the mouse, rat, and mouse-rat chimeras, including both hematopoietic and niche cells (endothelial cells, chondrocytes, fibroblasts and myofibroblasts) of mouse (GFP+) and rat origin (GFP-) (new Figure 4 and Figure 4 —figure supplement 1A-D). Furthermore, we provided bioinformatic analysis of receptor-ligand interactions and identified signaling pathways between hematopoietic progenitors and stromal cells in both the rat and mouse BM compartments of interspecies mouse-rat chimeras (new Figure 5 and Figure 5 —figure supplements 1 and 2). Such detailed studies have not been conducted in the bone marrow of any interspecies chimeras, significantly increasing the novelty of our manuscript.

What the study needs in order to stand out and make ideal use of the mouse-rat chimeric system is a developmental perspective of how the murine ESCs are converted or incorporated into the embryo in order to produce definitive murine HSCs from the hemogenic endothelium of dorsal aorta. Or alternatively a better characterization, functional or transcriptional of the fetal GFP+ murine HSCs during maturation in the fetal liver.

We agree. As the Reviewer suggested, we characterized the contribution of mouse ESCs to HSCs during embryonic development of mouse-rat chimeras by providing new immunostaining of dorsal aorta for RUNX1 and FLK1 (new Figure 6A) and FACS analysis of the fetal liver (new Figure 6B and Figure 6 —figure supplement 2). Based on co-expression of RUNX1 transcription factor and FLK1 (VEGF receptor 2), hemogenic endothelium in chimeric dorsal aorta develops earlier from donor mouse ESCs compared to endogenous rat ESCs (Figure 6A). Furthermore, we used FACS analysis of fetal E15.5 livers for CD117, Sca1, CD48 and CD150 in Lin cells to show an increase in the number of fetal murine hematopoietic progenitor cells (Figure 6B and Figure 6 —figure supplement 2). All these data are consistent with increased numbers of murine HSCs in the bone marrow of mouse-rat chimeras (Figure 4).

It would be very enlightening to determine if the murine tissue must transition directly from the hemogenic endothelium to functional HSCs or if it can emerge denovo from non-GFP+ hemogenic endothelium. Since the cells are GFP-labeled and clearly identifiable from the rat tissue, this analysis can be performed by flow cytometry, single-cell RNAseq or even imaging and microscopy. It is not known when and how the chimeric system produces definitive HSCs in the embryos and the system that investigators have utilized to study the adult bone marrow is capable of answering this important question and thus producing a truly novel study.

We agree. Based on new FACS and immunostaining data described above, it is likely that donor mouse ESC-derived hemogenic endothelium undergoes direct transition to functional HSCs in the fetal liver, whereas rat-derived (non-GFP+) hemogenic endothelium can be a source of rat HSCs. We acknowledged this possibility in the Discussion section (pages 14-15).

The average gestational time in C57Bl/6 mice is 19 days, whereas the gestational time in CD-1 rats is 21 days. Since mouse embryos develop faster compared to rat embryos by approximately 1.5 days (new references Farrington-Rock et al., 2008; Marcela et al., 2012; Takahashi and Osumi, 2005; Torres et al., 2008, and new Figure 6 —figure supplement 1), it is possible that mouse ESC-derived cells migrate faster and colonize the developing hematopoietic niches in the mouse-rat chimeras, leading to a “competing advantage” and preferential development of murine HSCs compared to rat HSCs. These data are consistent with increased numbers of mouse ESC-derived hematopoietic progenitors in the dorsal aorta, fetal liver and bone marrow of mouse-rat chimeras. Our data suggest that using donor ESCs from species with less gestational time in interspecies “bioreactors” can lead to larger quantities of ESC-derived hematopoietic progenitors in the chimeric bone marrow. We included this statement in the Discussion section (page 15).

The authors have done a commendable job characterizing the bone marrow contribution of the blastocyst complementation system in their mouse-rat chimaeras. Their results clearly show the amount and the expression profiles GFP+ murine HSCs and progenitors. However, on its own this characterization is not enough to merit publication as it is not mechanistically novel and makes use of previously established systems and only incrementally advances the field by using the chimeric system in an inter-species model.

The single cell RNAseq is informative and drives home the point that the murine ESCs produce the HSCs that are transcriptionally similar to mouse-mouse chimaeras, but the fact that the cells are capable of reconstitution in a transplant setting is already sufficient to establish the functional potential of the mouse HSCs in a rat chimeric bone marrow. Thus, a mechanistic and/or developmental approach is required to determine when and how the mouse ESCs produce the definitive HSCs in the chimeric fetus. This can already be performed with the system that the authors have in hand and only requires timed matings and chimeric embryo extraction and characterization of the fetal GFP+ cells either in the AGM region of the dorsal aorta during HSC emergence or the fetal liver during HSC expansion.

We agree. As suggested by the Reviewer we performed timed mating, chimeric embryo extraction, and characterization of the fetal GFP+ hemogenic endothelial cells in the dorsal aorta using immunostaining for RUNX1 and FLK1. We also performed FACS analysis of E15.5 livers for GFP+ HSCs. These data are provided in new Figure 6A-B and Figure 6 —figure supplement 2. Furthermore, we provided new single-cell RNA seq data of early postnatal (P5) bone marrow to characterize signaling pathways between stromal and hematopoietic compartments in donor (murine) and recipient (rat) cells in chimeric bone marrow (new Figures 4A-C and 5, and new Figure 4 —figure supplement 1A-D and Figure 5 —figure supplements 1 and 2). All these data were incorporated into the Results and Discussion sections of the revised manuscript (pages 9-11 and 14-15).

It would be very interesting to determine if the mouse HSCs emerge at the same rate as the rat ones or if there is a different selection during emergence and expansion in the fetal liver that produces the high frequency of GFP+ LT-HSCs seen in the bone marrow (Figure 1H). This integration and incorporation of the mouse cells in the chimeric system during embryo development would truly set the story apart and provide novel findings essential to our understanding of developmental hematopoiesis and chimeric model systems in general.

We agree. Our new data demonstrate that the mouse HSCs emerge faster compared to the rat ones in the dorsal aorta of mouse-rat chimeras (new Figure 6A). We also observed increased numbers of GFP+ murine ST-HSCs during expansion in the fetal E15.5 liver (new Figure 6 —figure supplement 2). In contrast, the number of LT-HSCs in the fetal liver was unchanged. It is possible that the expansion of murine LT-HSCs in mouse-rat chimeras occurs after E15.5, leading to the increased percentage of these cells in the chimeric bone marrow (Figure 1H). We hope that our new findings increase novelty of our manuscript and improve our understanding of developmental hematopoiesis in chimeric model system.

Specific comments:

In Fig,1, for both the legend and the text of the manuscript, the use of the term "control" and "mouse" is used interchangeably, but it is difficult to understand what it refers to. The comparison of a WT mouse to a GFP+ mouse-rat chimaera is not ideal as a control in this case because in a mouse presumably all the HSCs and progenitors are detectable, while in a mouse-rat chimaera only a subset of the cells are GFP+HSCs. This is very evident in Figure 1F where the "mouse" seems to have no GFP+ BM contribution in the BM while the chimaera has on average 25% contribution of GFP+ cells in the BM. It is not obvious why this is the case? A better control would be the assessment and comparison of the rat HSCs and progenitors in the mouse-rat BM of the chimeric mice.

We agree. We provided new single-cell RNAseq of P5 bone marrows in which we directly compared mouse and rat hematopoietic progenitors in the mouse-rat chimeras. We also compared these cells to “normal BM cells” of rats and mice of the same age (new Figures 4 and 5, new Figure 4 —figure supplement 1A-D, and Figure 5 —figure supplements 1 and 2). Different contributions of mouse and rat cells in the mouse-rat chimeras were provided in new Figure 4C of the revised manuscript.

Unfortunately, Flow cytometry cannot be used to distinguish between mouse and rat cells in the chimeras because rat-specific Abs (which do not recognize mouse antigens) to most of cell surface markers are not commercially available. We provide Figure 1 —figure supplement 1A-B to show that mouse-specific Abs used in Figure 1 recognize only mouse but not rat cell surface molecules.

In Figure 3 and Figure 4 how does the expression of the endogenous rat HSCs compare to that of the mouse HSCs in the chimaera. Presumably these two similar cell types from different species share a common bone marrow niche and thus are expected to be similar unless the presence of the mouse GFP+ HSCs and progenitors affects the endogenous rat cells.

We agree. New single-cell RNAseq dataset from P5 chimeric BM was used to address this question. We provided new Figure 4 —figure supplement 1A-D in which we compared gene expression of endogenous rat hematopoietic and stromal cells with that of the mouse ESC-derived cells in the mouse-rat chimeras. Expression signatures and potential ligand-receptor interactions in mouse and rat cells were remarkably similar (new Figure 4 —figure supplement 1A-D, new Figure 5, and Figure 5 —figure supplements 1 and 2).

Whole bone marrow transplants (with 500,000 GFP+ cells) are not the ideal means to establish cell autonomous functionality of the HSCs. For this purpose, phenotypic GFP+ HSCs, as shown in Figure 1E, should be sorted and transplanted into irradiated donors. Ideally, the transplant should be performed once again in secondary recipients to fully establish the functional potential and self-renewal capacity of the chimeric HSCs.

As suggested by the Reviewer, we have performed this experiment by transplanting FACS-sorted, GFP+ HSCs (ST-HSC/ LT-HSC cell mixture) into irradiated recipient mice. The average number of transplanted GFP+ HSCs per mouse was 194 cells. In our experimental conditions, this number of HSCs was insufficient to rescue lethally irradiated mice, possibly, due to requirements for other progenitor and differentiated hematopoietic cells in the bone marrow to replace the loss of vast majority of white blood cells in the acute stage after the high-dose irradiation (Figure 8B, please see WBC numbers in the peripheral blood 8 days after irradiation).

To establish the functional potential and self-renewal capacity of the chimeric GFP+ HSCs, we performed the second bone marrow transplantation in secondary recipients and these new data are provided in Figure 8 —figure supplement 6A-E. The secondary transplantation of mouse ESC-derived bone marrow from the mouse-rat chimeras rescued irradiated mice (new Figure 8 —figure supplement 6B-C) and resulted in long-term engraftment into hematopoietic cell lineages of the peripheral blood and bone marrow (new Figure 8 —figure supplement 6D-E). These new data were incorporated into the revised manuscript (page 13).

Figure 6A is not necessary since it is entirely subjective and has no values listed in any of the chosen gates. It should be moved to the supplement and Figure 5 should then be combined with Figure 6.

We agree. As suggested by the Reviewer, we moved old Figure 6A to the supplement (new Figure 8 —figure supplement 1) and then combined the remaining panels of old Figures 6 and 7 into new Figure 8.

Figure 7A is not informative and should be removed from the manuscript. Instead, the authors should perform longitudinal sections of the bone (femur) and stain for H and E to show the effects of IR on the bone marrow microenvironment in the listed timepoints and treatments.

We agree. Figure 7A was removed from main figures and placed in the supplement (new Figure 8 —figure supplement 4). As requested by the Reviewer, we provided H and E-stained longitudinal sections of the femur in new Figure 7D. We also provided GFP images of frozen BM tissue sections to demonstrate the abundance of mouse ESC-derived (GFP+) cells in the BM compartment of transplanted mice (new Figure 7D, bottom panels).

Reviewer #3 (Recommendations for the authors):

Rat-mouse interspecies chimeras have been used for many years and are useful to study the impact of various gene deficiencies in reconstituting different organs or tissues by mixing mutated and wild type cells. Here the authors have focused on the bone marrow and more particularly on the hematopoietic compartment.

The paper by Weng et al., aims at investigating the formation of the bone marrow with a focus on the hematopoietic compartment. They are using rat-mouse interspecies chimeras where mouse embryonic stem cells GFP-tagged are injected into rat blastocysts that subsequently develop into viable chimeric animals after reimplantation into pseudo-pregnant females. The authors sort the bone marrow cells from the chimeras on the basis of GFP and analyzed the hematopoietic cell populations with the standard flow cytometry approaches and reconstitution analysis in irradiated recipients. They conclude that a mouse-derived complete and functional hematopoietic hierarchy is present in the chimeric animals with an increase of short-term and long-term mouse hematopoietic stem cells compared to non-injected mice.

They analyzed the hematopoietic cell populations using single cell RNA sequencing and found weak or no difference compared to non-chimeric animals and they probed the reconstitution potential of the hematopoietic stem cell compartment.

We would like to thank the Reviewer for summarizing the results and conclusions of our manuscript.

This is an interesting study that is essentially descriptive regarding the system of chimerism. The authors have however biased the analysis by focusing on the hematopoietic compartment and not on the entire bone marrow as they claim. Of note, the significant increase in the hematopoietic stem cell compartment observed in the chimeric context has not been analyzed. This is a key point of the study since it may help isolating factors to amplify hematopoietic stem cells. Thus despite an interesting design, this study falls short in finding key mechanisms of hematopoietic stem cell homeostasis and self-renewal.

Based on constructive comments of the reviewers, we provided additional experimental data in new Figures 4, 5, 6, and 7D, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2, Figure 6 —figure supplements 1A-C and 2, Figure 8 —figure supplement 6A-E, which characterize both hematopoietic and stromal cellular components in the chimeric bone marrow and analyze receptor-ligand interactions between hematopoietic and stromal cells in the chimeras. Our new studies identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44 and App-Il18rap signaling pathways between hematopoietic progenitors and stromal cells (new Figure 5 and Figure 5 —figure supplements 1 and 2). It is possible that modulating these pathways may help to amplify hematopoietic progenitor cells in the interspecies bone marrow. We also performed additional studies to characterize the development of donor hematopoietic progenitor cells during embryonic and early neonatal (P5) time points using the bone marrow, dorsal aorta, and fetal livers from mouse-rat chimeras (new Figures 4, 5 and 6, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2, Figure 6 —figure supplements 1A-C and 2). Altogether, our new data provide important information about signaling and developmental mechanisms that lead to formation of the BM compartment in interspecies chimeras.

Strengths

Exploring alternative approaches for the production of transplantable hematopoietic stem cells for therapeutic purposes.

Mastering interspecies rat-mouse chimeras to study the functioning of species-specific hematopoietic systems in a mixed bone marrow environment.

We would like to thank the Reviewer for summarizing key strengths of our manuscript.

Weaknesses

Short, mostly descriptive study with few insights into how to improve hematopoietic stem cell manipulation and amplification.

Bias in the analysis by using only the hematopoietic fraction of the GFP+ population. The authors claim they are studying the bone marrow but they are in fact analyzing the hematopoietic compartment. The stromal cell compartment is totally lacking.

We performed additional experiments and provided new experimental data in which we used single-cell RNAseq to characterize both hematopoietic and stromal cellular components in the chimeric bone marrow (new Figures 4 and 5, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2).

The single cell approach is biased since authors are artificially mixing different percentages of lin+ and lin- cells. The most interesting part of the work i.e., the amplification of short-term and long-term hematopoietic stem cells in chimeras should not be approached this way.

We would also like to point out that the mixing of different cell types was done because we followed the previously published single cell RNAseq protocol (Baccin et al., 2020, Nat Cell Biol), which allows to obtain sufficient numbers of rare hematopoietic progenitor cells for bioinformatic analysis.

In the revised manuscript, we provided new single-cell RNA sequencing datasets which were produced without mixing of Lin+ and Lin cells (new Figures 4 and 5, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2). These new datasets were integrated with the old single cell RNAseq data in the revised manuscript.

The long term reconstitution lacks secondary transplantation to show that hematopoietic stem cells are endowed with long-term reconstitution potential.

We agree. As requested by the Reviewer, we performed the secondary bone marrow transplantation and found that mouse ESC-derived hematopoietic stem cells are endowed with long-term reconstitution potential (new Figure 8 —figure supplement 6A-E).

Reviewer #3 (Recommendations for the authors):

This is an interesting study that might give clues into how create and/or amplify hematopoietic stem cells.

Please focus on the entire bone marrow rather than on the hematopoietic cell compartment.

We agree. We provided additional experimental data in new Figures 4 and 5, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2 that characterize both hematopoietic and stromal cellular components in the chimeric bone marrow.

Analyze the transcriptome without introducing bias. The idea is to understand how the hematopoietic stem cell compartment is expanded in chimeras, not to show that all the hematopoietic cell lineages are present.

We agree. We provided new single-cell RNAseq datasets of the bone marrow without mixing of Lin+ and Lin cells (new Figures 4 and 5, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2) which were integrated in the revised manuscript. We also performed the receptor-ligand analysis between hematopoietic and stromal cells to highlight the potential significance of Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44 and App-Il18rap signaling pathways in expansion of hematopoietic progenitor cells in interspecies chimeras. Finally, we performed analysis of hemogenic endothelium and HSCs in the dorsal aorta and fetal liver and found that mouse cells faster colonize the developing hematopoietic niches in the mouse-rat chimeras (new Figure 6A-B and Figure 6 —figure supplements 1A-C and 2), possibly, leading to a “competing advantage” and preferential development of murine HSCs compared to rat HSCs.

Analyze the stromal cell compartment that is complementary to the hematopoietic stem cell compartment.

We agree. The analysis of the stromal BM compartment was performed using single-cell RNAseq. These data were included in the revised manuscript (new Figures 4 and 5, and Figure 4 —figure supplement 1A-D, Figure 5 —figure supplements 1 and 2).

Perform secondary transplantation to show that secondary reconstitution can take place.

We agree. We provided these data in new Figure 8 —figure supplement 6A-E.

Expand your analysis to cytokines and growth factors.

We agree. We used receptor-ligand analysis between hematopoietic and stromal cells to identify several signaling molecules and their receptors which can be involved in expansion of mouse hematopoietic progenitor cells in interspecies chimeras (new Figure 5 and Figure 5 —figure supplements 1 and 2).

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

We would like to thank the Editor for handling the review of our manuscript and for providing constructive comments.

1. The concern raised by Reviewer 2 about the functional potential of the GFP+ mouse BM HSCs from the chimeric mice.

We addressed the concern of the Reviewer 2 by modifying our manuscript to avoid statements that “mouse HSCs are fully functional” in our experimental setting. Instead, we used the words “the bone marrow from mouse-rat chimeras was functional”. This statement is fully supported by our studies because (1) the bone marrow from mouse-rat chimers rescued lethally irradiated mice and restored all hematopoietic cell lineages in the peripheral blood and bone marrow after the primary and secondary transplantation; and (2) the bone marrow from mouse-rat chimers contains all ESC-derived hematopoietic progenitors and stromal cell subsets that were detected in normal mice and rats.

Furthermore, we modified the Discussion section to acknowledge a limitation of our study about HSC functional potential as follows (page 16):

“…One of the limitations of our studies is that the functional potential of chimeric HSCs was established from whole BM transplants and not from transplantation of purified HSCs. While these experiments are technically challenging, transplantation of FACS-sorted donor HSCs into lethally irradiated mice will be needed in our future studies to investigate whether chimeric HSCs are fully functional to restore all hematopoietic cell lineages after irradiation.”

2. Addressing the comments of Reviewer 3 about the presentation of specific figure panels to illustrate the key points of the study in a cohesive manner and the need for sufficient rationale + context.

We agree. As suggested by the Reviewer 3, we moved Figure 3 into the supplement, provided low magnification images of embryos in Figure 5A, replaced several flow cytometry panels in Figure 1E, provided new Figure 1I showing the total numbers of HSCs in chimeras, and included additional rationale and experimental details for single cell RNA-seq experiments. Specific points are addressed below in the answer to reviewer’s critique.

It is likely that the questions can be addressed with existing data which needs to be prioritized (main figures versus supplement), presented in a cohesive manner, elaborated (experimental details) on and discussed more extensively (whenever there are limitations which affect the conclusions).

We agree. Based on reviewer’s comments, we revised the presentation of experimental data in the main figures and supplements (main Figures1, 2 and 5, and Figure 2 —figure supplements 5, 6 and 7), provided additional experimental details in the Methods section (pages 18-20) and provided an additional discussion about limitations of our studies as related to transplantation of purified HSCs to irradiated mice (page 16).

Reviewer #1 (Recommendations for the authors):

The authors have substantially expanded the manuscript through highly relevant new studies in response to the reviewer comments and have thereby increased the robustness and significance of their findings.

The significant improvements include the addition of more time points, the analysis of putative ligand receptor interactions and the secondary bone marrow transplantation studies.

The scRNA-seq data have been uploaded to GEO and are accessible to the public.

We would like to thank the Reviewer for summarizing the improvements in our revised manuscript and for acknowledging the robustness and significance of our findings.

Reviewer #2 (Recommendations for the authors):

Overall manuscript is much improved, has broader appeal and now provides very intriguing findings on the developmental perspective of when HSCs emerge in a chimeric embryo as a potential timing regulation of LT-HSCs expansion in the fetal liver.

The authors were mostly responsive to the critiques and have sufficiently improved the novelty of the manuscript by performing experiments in the developing embryo.

We would like to thank the Reviewer for acknowledging the improvements and novelty of our revised manuscript.

However, there is one remaining critical concern regarding the functional potential of the GFP+ mouse BM HSCs from the chimaera. The authors say that they attempted to transplant sorted GFP+ stem cells but only about 198 LT-HSCs and ST-HSC were purified on average per chimaera BM. Furthermore, they say that these cells didn't provide enough hematopoietic support for a viable recipient mouse. This is not a valid response and raises a very big question about the cell intrinsic functional potential of the GFP+ mouse BM HSCs. Considering that 500,000 total cells provide reconstitution even long-term secondary transplant reconstitution, and with Figure 1H showing that there are chimaeras with as many as 40% GFP+ ST-HSCs and LT-HSCs, the ability to conduct a transplant with purified HSCs should be possible.

We understand the Reviewer concern and we have tried to perform this experiment during the revision process which took approximately 9 months. Such experiments are time-consuming because the chimeras must be created before the transplantation. It is likely, that our inability to rescue lethally irradiated mice with FACS-sorted GFP+ HSCs can be explained by technical difficulties. Since HSC are rare, a long time of cell sorting can lead to decreased cell viability which directly affects the transplantation potential of HSCs. As the Reviewer noted, our Figure 1H shows that some chimeras have as many as 40% HSCs. However, this number of HSCs is among LSKs that account for minor percentage of Lin-negative cells in the bone marrow (Figure 1E and 1G). Considering that Lin-negative cells represent only 5% of GFP+ (ESC-derived) cells (Figure 1E), the overall number HSCs in the bone marrow is still low. The long time of FACS sorting can explain our technical difficulties with the requested experiments.

The authors did not have to test the average of GFP+ HSCs per chimaera, they simply had to show that some of the GFP+ cells were capable of cell autonomous long-term reconstitution. This brings to question the absolute number of mouse GFP+ HSCs in the chimaera bone marrow. Which should be determined from their flow cytometric analysis.

As requested by the Reviewer, we provided new Figure 1I which shows the absolute number of mouse GFP+ HSCs in the chimeric bone marrow determined by flow cytometric analysis. These new data were integrated in the Results section of the revised manuscript (pages 6-7).

The authors have to explain how they purified the ST- and LT-HSCs from the bone marrow and why 198 could not provide them with a viable recipient mouse when work from the Weissman and Morrison labs have shown that as few as 10 LT-HSCs (SLAM LSKs) can provide functional hematopoiesis in a transplant setting. Did the authors support the recipient mice with lineage+ splenocytes? What was the dose of irradiation administered? There has to be a better description of this experiment in the methods, in fact the methods as listed in the revision are very light on details of transplantation and lineage analysis.

We don’t doubt the high-quality experimental data from the Weissman and Morrison labs. As we explained above, this experiment didn’t work in our experimental settings due to, most likely, technical reasons which can include the long time of FACS sorting and/or insufficient support from lineage+ splenocytes among other reasons.

We would also like to point out, that the overall goal of our study was to produce a functional bone marrow (as an organ) from mouse ESCs in a rat, which was achieved using blastocyst complementation with subsequent analysis of the bone marrow by Flow cytometry, single cell RNA sequencing, and primary and secondary bone marrow transplantations into lethally irradiated mice. Testing functionality of every hematopoietic and stromal cell type in the bone marrow, even such important cells as HSCs, was not in the scope of this study but will be a subject of future research.

Because this experiment didn’t work, we don’t think that description of this experiment is needed in the Materials and methods section. However, we have added additional details of bone marrow transplantation and lineage analysis to the Methods section (pages 18 and 20) and discussed the limitation of our study about HSC functionality in the Discussion section (page 16).

So while the functional potential of chimeric stem cells has been established from the bulk bone marrow transplants, the lack of purified GFP+ mouse HSCs transplants limits the conclusions that the authors make with regard to calling these mouse HSCs functional in their experimental setting.

We understand the Reviewer concern that such experiment is needed to establish the functionality of HSCs. Therefore, we modified our manuscript to avoid statements that “the chimeric HSCs are fully functional” in our experimental setting. Instead, we used the words “the bone marrow from mouse-rat chimeras is functional”. This statement is fully supported by our studies because (1) the bone marrow from mouse-rat chimers rescued lethally irradiated mice and restored all hematopoietic cell lineages in the peripheral blood and bone marrow, including HSCs, after the primary and secondary transplantation; and (2) the bone marrow from mouse-rat chimers contained all ESC-derived hematopoietic progenitors and stromal cell subsets that were detected in normal mice and rats under our experimental conditions using the single cell RNA sequencing method.

Furthermore, we modified the Discussion section to acknowledge a limitation of our study about HSC functionality as follows (page 16):

“One of limitations of our studies is that the functional potential of chimeric HSCs was established from the whole BM transplants but not from transplantation of purified HSCs. While these experiments are technically challenging, transplantation of FACS-sorted donor HSCs into lethally irradiated mice will be needed in our future studies to investigate whether chimeric HSCs are fully functional to restore all hematopoietic cell lineages after irradiation.”

Reviewer #3 (Recommendations for the authors):

This reviewer thanks the authors for the revised version of their manuscript that shows significant improvement.

On the basis of this revised version, you will find enclosed my main comments for manuscript improvement. In general, I have the feeling that some of the figures are redundant or that the authors have trouble making a choice on what they want to show.

We would like to thank the Reviewer for additional comments and valuable suggestions for improvements of our revised manuscript.

Figure 1: The authors examine the percentages of the ST-HSC and LT-HSC in WT mice vs chimeric animals. Ideally, a mouse/mouse chimeric control should also be included. In the Sca-Kit analysis (Fig1E); the authors mentioned a significant increase in the Sca-Kit compartment in chimeras vs WT animals. This is substantiated in Fig1G, although two groups of animals with distinct (high and low) percentages of Sca-Kit are visible but not substantiated in Figure 1E where the authors show close percentages between the two conditions. The same remark applies in the opposite direction for the percentages of LT vs ST HSC. I would recommend more carefully choosing the panels to illustrate their conclusions. The rationale of analyzing the animals at P10 is rather uncommon in hematology unless you want to analyze the forming bone marrow. P30 would have been more classical.

We apologize for the lack of clarity in some Figure 1E panels. As suggested by the Reviewer, we revised our manuscript to replace Flow cytometry panels of the Sca-Kit analysis (Figure 1E). New Figure 1E panels are consistent with quantification of the data (Figure 1G-I). We would like to point out that our manuscript has a developmental focus since we analyzed embryos (E11-15) and postnatal time points (P5 and P10), providing a rationale for using P10 chimeras instead of more classical P30 time-point in our studies. The mouse control was provided in Figure 1E-I to show percentages of bone marrow cells in comparison with mouse-rat chimeras of same age.

Figure 2: The design of this experiment is still puzzling for me and is not clearly indicated either in the text or in the legend. The authors have sorted GFP+ cells from the BM. Why is it necessary to enrich in committed hematopoietic progenitors since the BM already contains high numbers of these cells? Please clarify.

We agree. We provided additional rationale for single cell RNAseq experiment in the Results section (page 7) and the Methods section (pages 18-19). Since the numbers of HSCs and other hematopoietic progenitor cells in the bone marrow are significantly low compared to numbers of differentiated hematopoietic cells, we enriched for BM progenitor cell populations prior to single cell RNA sequencing by combining 90% of FACS-sorted GFP+Lin cells and 10% of GFP+Lin+ cells in each experimental group. This enrichment enabled us to obtain enough progenitor cells for UMAP clustering analysis.

The rationale of mixing mouse/mouse and mouse/rat GFP+ cells is not clear since the authors do not identify cells originating from one combination vs the other.

We apologize for the confusion. We performed single cell RNAseq experiments separately for mouse-mouse and mouse-rat GFP+ cells. We made it clear in the revised Methods section (pages 18-19). We also removed old Figure 2A which created this confusion because in this figure both cell types were combined to show that mouse-mouse cells were indistinguishable from mouse-rat cells based on gene expression signatures. Revised Figure 2A show bioinformatic analysis of GFP+ cells separately for mouse-mouse and mouse-rat chimeras.

At this point, instead of performing a scRNA-seq, a GFP+ FACS sorting followed by a multilineage hematopoietic analysis by flow cytometry i.e.myeloid, lymphoid, erythroid cells would have been more simple and direct. Could you please clarify the design of the experiment?

We provided multi-lineage hematopoietic analysis of the bone marrow for Lin-negative, LSKs, ST-HSCs and LT-HSCs in Figure 1E-I and described it in the revised Methods section (page 20). The advantage of using single cell RNAseq is that many more cell types can be identified compared to FACS analysis. The design of scRNAseq experiment in Figure 2 was clarified in the revised Results section (page 7) and in the Methods section (pages 18-19).

More generally the authors strongly emphasize on the transcriptome analysis. Several main figures are redundant and could be switched to supplementary ones e.g. Figure 3 and Figure 4 since these latter does not bring any specific insights into species-specific mechanisms.

As the Reviewer suggested, we moved old Figure 3 into Supplemental material (new Figure 2 —figure supplement 5). Based on our data presentation, we cannot move old Figure 4 (new Figure 3) into supplemental material because this figure contains new single cell RNAseq experiment which shows both hematopoietic and stromal cells in the bone barrow. This experiment was requested in the first revision and provides an important support for the next main figure 4 and multiple supplemental figures that bring specific insights into species-specific mechanisms.

Fig6 should be revised. The sections of the embryonic aorta are not informative regarding the endothelium and the hematopoietic cell production. Please provide images showing low magnification of the aorta and the surrounding structures and high magnification of the hematopoietic clusters.

We agree. As requested by the Reviewer, we provided additional images showing low magnification of the dorsal aorta and the surrounding structures (new Figure 5A). High magnification of hemogenic endothelium and hematopoietic clusters is provided in Figure 5B.

Fig8: Multilineage analysis of the blood is interesting but the most informative piece of data comes from the BM analysis. Showing results at 8 days post-irradiation is not very informative since the animals are in aplasia.

We provided multilineage analyses of both BM and blood in main Figure 7 (old Figure 8) and six supplements to the Figure 7 (Figure 7 —figure supplements 1-6). The data were arranged to provide the most important findings in main Figure 7. The rest of the data were moved to supplements. Since we already obtained results at 8 days post-irradiation, we believe it is important to provide the readers this information and help them evaluating the aplasia and rescue experiments.

https://doi.org/10.7554/eLife.74018.sa2

Article and author information

Author details

  1. Bingqiang Wen

    Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8827-4820
  2. Guolun Wang

    Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Enhong Li

    Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Data curation, Formal analysis, Validation, Visualization
    Competing interests
    No competing interests declared
  4. Olena A Kolesnichenko

    Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Resources, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Zhaowei Tu

    Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Senad Divanovic

    1. Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, United States
    2. Department of Pediatrics, College of Medicine of the University of Cincinnati, Cincinnati, United States
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7538-0499
  7. Tanya V Kalin

    1. Department of Pediatrics, College of Medicine of the University of Cincinnati, Cincinnati, United States
    2. Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Resources, Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Vladimir V Kalinichenko

    1. Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    2. Department of Pediatrics, College of Medicine of the University of Cincinnati, Cincinnati, United States
    3. Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    4. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
    Contribution
    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    Vladimir.Kalinichenko@cchmc.org
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3438-2660

Funding

National Heart, Lung, and Blood Institute (HL141174)

  • Vladimir V Kalinichenko

National Heart, Lung, and Blood Institute (HL149631)

  • Vladimir V Kalinichenko

National Heart, Lung, and Blood Institute (HL152973)

  • Vladimir V Kalinichenko

National Heart, Lung, and Blood Institute (HL158659)

  • Tanya V Kalin

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Mrs. Erika Smith for excellent secretarial support. This work was supported by NIH Grants HL141174 (to VVK), HL149631 (to VVK), and HL152973 (to VVK and TVK).

Ethics

All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Cincinnati Children's Research Foundation (protocol # IACUC2016-0038).

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Jalees Rehman, University of Illinois at Chicago, United States

Reviewer

  1. Jalees Rehman, University of Illinois at Chicago, United States

Publication history

  1. Received: September 18, 2021
  2. Preprint posted: October 1, 2021 (view preprint)
  3. Accepted: September 29, 2022
  4. Accepted Manuscript published: September 30, 2022 (version 1)
  5. Version of Record published: October 18, 2022 (version 2)

Copyright

© 2022, Wen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Bingqiang Wen
  2. Guolun Wang
  3. Enhong Li
  4. Olena A Kolesnichenko
  5. Zhaowei Tu
  6. Senad Divanovic
  7. Tanya V Kalin
  8. Vladimir V Kalinichenko
(2022)
In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras
eLife 11:e74018.
https://doi.org/10.7554/eLife.74018

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