- Email: [email protected]
Tracking HSC Origin: From Bench to Placenta
Developmental Cell
Previews Tracking HSC Origin: From Bench to Placenta Vincenzo Calvanese1,2 and Hanna K.A. Mikkola1,2,3,* 1Department
of Molecular, Cell and Developmental Biology and Edythe Broad Center for Regenerative Medicine and Stem Cell Research 3Molecular Biology Institute University of California Los Angeles, Los Angeles, CA 90095, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.02.022 2Eli
Reporting in Developmental Cell, Pereira et al. (2016) use in vitro lineage reprogramming insights to inform understanding of hematopoietic stem cell (HSC) development in vivo. They find Prom1+Sca1+CD34+CD45 hemogenic precursors, akin to fibroblast-derived hemato-vascular precursors, in mouse placenta and embryo. The cells mature into transplantable HSCs in culture. Lineage reprogramming with tissuespecific transcription factors is one of the most revolutionary approaches to generate patient-specific stem/progenitor cells for cell-based therapies. Several reports have witnessed that by selecting factor combinations that govern the identity and function of the desired cell type, it is possible to break the epigenetic boundaries between cell types and ‘‘transdifferentiate’’ almost any cell to another lineage, including blood, neuron, pancreatic islets, and heart muscle (Jopling et al., 2011). However, a major question is whether the cells created by lineage reprogramming have real in vivo counterparts or whether they represent unorthodox cell types that have only partial characteristics of the desired cell and are functionally incomplete. This question has been difficult to answer, as often the precise surface phenotype and molecular profile of the target cell is unknown. Hematopoietic stem cells (HSCs) are an attractive target for lineage reprogramming because they have proven their value in curing many serious blood diseases. The major limitation preventing broader use of HSCs is the lack of HLA (human leukocyte antigen)-matched HSC donors, which could be overcome if HSCs could be lineage-reprogrammed directly from the patient’s cells or differentiated from patient-specific induced pluripotent stem cells. To succeed in generating HSCs using such approaches, we need a better understanding of how hemogenic precursors become selfrenewing HSCs. However, the dynamic nature of developmental hematopoiesis makes it challenging to study HSCs and their precursors, because hematopoiesis
switches between multiple anatomical sites and because the surface markers to identify cells of the HSC lineage change as development progresses. Although it is now experimentally proven that HSCs derive from endothelial precursors, not all endothelia are hemogenic, and not all hemogenic endothelia generate HSCs. A particular challenge in the field has been to identify markers for the precursors that generate true HSCs rather than short-lived embryonic progenitors, which are generated prior to the self-renewing HSCs and rapidly populate all hemogenic organs through circulation. Moreover, the precise anatomical origin of HSCs also remains a topic of discussion. Many investigators still consider the aortagonad-mesonephros region (AGM) as the sole or main site of HSC generation, although several studies have provided evidence that the placenta and the yolk sac also participate in HSC development (Chen et al., 2009; Gekas et al., 2005; Ottersbach and Dzierzak, 2005; Rhodes et al., 2008). In this issue of Developmental Cell, Pereira et al. (2016) take advantage of their discoveries from in vitro lineage reprogramming studies to define an immediate precursor to mouse HSCs and define their anatomical localization in the mouse conceptus (Figure 1). In an earlier seminal work in 2013, Pereira and colleagues described directed lineage reprogramming of mouse embryonic fibroblasts to hematopoietic stem/progenitor cells (HSPCs) by overexpressing four factors highly expressed in long-term reconstituting HSCs: Gata2, Gfi1b, cFos, and Etv6 (Pereira et al., 2013). Using this approach, they generated early hemogenic precur-
sors (HPs) that expressed Sca1, CD34, and Prom1 (herein called PS34 cells) but were devoid of the pan-hematopoietic marker CD45 expression. Sca1 and CD34 are established markers of HSCs during mouse development, and Prom1 (Prominin1/CD133) has been used as a HSPC marker in humans (Wirthlin et al., 2008). In their new study, Pereira et al. (2016) now identify HP populations with similar PS34 surface phenotype in the developing mouse conceptus and confirm that PS34 cells can be retrieved in multiple sites of HSC development, including the placenta, the AGM region, vitelline, and umbilical vessels (Figure 1). Importantly, the authors show that placental PS34 cells are fetal rather than maternal in origin. This study thus represents a major advance in pinpointing the anatomical origin of definitive HSCs, as it confirms that the placenta is not only a nursery for HSCs during midgestation but also a site of HSC emergence. Future studies will be required to assess whether the yolk sac, another disputed site of HSC development, harbors PS34 HSC precursors. Molecular profiling of placental PS34 cells both at the population and singlecell levels confirmed several expected characteristics of HSC precursors. These include endothelial identity with molecular features of arterial endothelium, as well as the expression of the early endothelial-tohematopoietic transition factors, but the absence of genes expressed in more mature hematopoietic cells. Interestingly, PS34 cells from the placenta clustered together with hemogenic cells obtained by lineage reprogramming at 20 days. Moreover, placental PS34 cells expressed
Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc. 479
Developmental Cell
Previews Etv6 cFos Gata2
Gfi1b
Prom1+Sca1+CD34+ (PS34) cells
fibroblasts
AFT024 co-culture
Placenta PS34
OP9-Dll1 co-culture
HSPC phenotype Colony forming potential
Re-aggregate Placenta co-culture
HSC maturation
BM engraftment
E10.5 Prom1 YFP Lineage tracing AGM
BM seeding
E10.5-12.5 mouse embryo
Figure 1. Lineage Reprogramming of Fibroblasts to Hematopoietic Cells Uncovers the Identity for Hemogenic Precursors that Generates Definitive HSCs in the Placenta and the AGM Top: Enforced expression of four transcription factors (Gata2, Etv6, Gfi1b, and cFos) in mouse embryonic fibroblasts followed by conditioning on hematopoiesis-supportive stroma (AFT024) drives the generation of hemogenic precursors (HPs) with Prom1+Sca1+CD34+ (PS34) surface phenotype. PS34 cells possess the potential to generate HSPCs that produce hematopoietic colonies upon downregulation of the reprogramming factors. Middle: Cells with PS34 phenotype were identified in E10.5–E12.5 mouse conceptus in the placenta vascular labyrinth and in the aorta-gonad-mesonephros region and adjacent arteries. PS34 cells were able to undergo maturation to HSCs in a Notch1-stimulating stroma (OP9-Dll1) and engraft in irradiated mouse recipients. Bottom: Lineage tracing using Prom1-dependent Cre activation at E10.5 labels Prom1-expressing cells with YFP (yellow). These cells are traced into the adult bone marrow (BM) HSCs and their progeny, confirming that Prom1 expression marks definitive HSC precursors.
high levels of Gata2, cFos, and Etv6, the key factors used in hematopoietic lineage reprogramming, and also upregulated the fourth reprogramming factor, Gfi1b, as they transitioned to HSPCs. The authors used state-of-the-art functional assays to show that although PS34 HPs isolated directly from the placenta did not have clonogenic potential, they matured into transplantable HSCs when co-cultured on OP9-Dll1 stroma that induced Notch1 signaling. Secondary transplantation confirmed that these cells were bona fide HSCs. The discovery that PS34 HPs rely on a Notch1-dependent maturation step in culture to acquire engraftment ability is an important advance. While Notch1 is known to be necessary in hemogenic endothelium for proper HSC development (Butko et al.,
2016; Kumano et al., 2003), this study shows that Notch1 signaling is also sufficient to convert early hemogenic precursors to transplantable HSCs in culture. This finding may help improve in vitro protocols for successful HSC generation. Finally, lineage tracing of cells that expressed Prom1 around E10.5 of mouse development verified their contribution to the adult hematopoietic system even in the absence of additional in vitro steps. This study thus provides an incredibly thorough characterization of the immediate HSC precursor at the phenotypic, molecular, and functional levels and offers new approaches to study how HSC precursors mature into functional HSCs. The discovery that PS34 cells generated in vitro through lineage reprogramming have a very similar in vivo counter-
480 Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc.
part will lead to interesting future studies comparing the similarities and differences between in-vitro-reprogrammed and invivo-derived PS34 cells, which may help identify the missing factors required to obtain engraftable HSCs from lineage-reprogrammed PS34 cells. Importantly, this study shows that the lessons learned from transcription factor-mediated lineage reprogramming are not only useful for engineering cells for regenerative medicine but can also be informative on developmental processes that occur during embryogenesis, which would be otherwise difficult to capture due to the dynamic nature and complexity of these systems. The ability to identify the hemogenic precursors that are on track to become self-renewing HSCs, either in vivo or in vitro, is a major advance in the field and paves the way for establishing protocols to generate HSCs for transplantation therapies.
REFERENCES Butko, E., Pouget, C., and Traver, D. (2016). Dev. Biol. 409, 129–138. Chen, M.J., Yokomizo, T., Zeigler, B.M., Dzierzak, E., and Speck, N.A. (2009). Nature 457, 887–891. Gekas, C., Dieterlen-Lie`vre, F., Orkin, S.H., and Mikkola, H.K. (2005). Dev. Cell 8, 365–375. Jopling, C., Boue, S., and Izpisua Belmonte, J.C. (2011). Nat. Rev. Mol. Cell Biol. 12, 79–89. Kumano, K., Chiba, S., Kunisato, A., Sata, M., Saito, T., Nakagami-Yamaguchi, E., Yamaguchi, T., Masuda, S., Shimizu, K., Takahashi, T., et al. (2003). Immunity 18, 699–711. Ottersbach, K., and Dzierzak, E. (2005). Dev. Cell 8, 377–387. Pereira, C.F., Chang, B., Qiu, J., Niu, X., Papatsenko, D., Hendry, C.E., Clark, N.R., Nomura-Kitabayashi, A., Kovacic, J.C., Ma’ayan, A., et al. (2013). Cell Stem Cell 13, 205–218. Pereira, C.-F., Chang, B., Gomes, A., Bernitz, J., Papatsenko, D., Niu, X., Swiers, G., Azzoni, E., de Bruijn, M.F.T.R., Schaniel, C., et al. (2016). Dev. Cell 36, this issue, 525–539. Rhodes, K.E., Gekas, C., Wang, Y., Lux, C.T., Francis, C.S., Chan, D.N., Conway, S., Orkin, S.H., Yoder, M.C., and Mikkola, H.K. (2008). Cell Stem Cell 2, 252–263. Wirthlin, L., Hess, D., Zhou, P., and Nolta, J. (2008). Biol. Blood Marrow Transplant. 14, 151–153.
Previews Tracking HSC Origin: From Bench to Placenta Vincenzo Calvanese1,2 and Hanna K.A. Mikkola1,2,3,* 1Department
of Molecular, Cell and Developmental Biology and Edythe Broad Center for Regenerative Medicine and Stem Cell Research 3Molecular Biology Institute University of California Los Angeles, Los Angeles, CA 90095, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.02.022 2Eli
Reporting in Developmental Cell, Pereira et al. (2016) use in vitro lineage reprogramming insights to inform understanding of hematopoietic stem cell (HSC) development in vivo. They find Prom1+Sca1+CD34+CD45 hemogenic precursors, akin to fibroblast-derived hemato-vascular precursors, in mouse placenta and embryo. The cells mature into transplantable HSCs in culture. Lineage reprogramming with tissuespecific transcription factors is one of the most revolutionary approaches to generate patient-specific stem/progenitor cells for cell-based therapies. Several reports have witnessed that by selecting factor combinations that govern the identity and function of the desired cell type, it is possible to break the epigenetic boundaries between cell types and ‘‘transdifferentiate’’ almost any cell to another lineage, including blood, neuron, pancreatic islets, and heart muscle (Jopling et al., 2011). However, a major question is whether the cells created by lineage reprogramming have real in vivo counterparts or whether they represent unorthodox cell types that have only partial characteristics of the desired cell and are functionally incomplete. This question has been difficult to answer, as often the precise surface phenotype and molecular profile of the target cell is unknown. Hematopoietic stem cells (HSCs) are an attractive target for lineage reprogramming because they have proven their value in curing many serious blood diseases. The major limitation preventing broader use of HSCs is the lack of HLA (human leukocyte antigen)-matched HSC donors, which could be overcome if HSCs could be lineage-reprogrammed directly from the patient’s cells or differentiated from patient-specific induced pluripotent stem cells. To succeed in generating HSCs using such approaches, we need a better understanding of how hemogenic precursors become selfrenewing HSCs. However, the dynamic nature of developmental hematopoiesis makes it challenging to study HSCs and their precursors, because hematopoiesis
switches between multiple anatomical sites and because the surface markers to identify cells of the HSC lineage change as development progresses. Although it is now experimentally proven that HSCs derive from endothelial precursors, not all endothelia are hemogenic, and not all hemogenic endothelia generate HSCs. A particular challenge in the field has been to identify markers for the precursors that generate true HSCs rather than short-lived embryonic progenitors, which are generated prior to the self-renewing HSCs and rapidly populate all hemogenic organs through circulation. Moreover, the precise anatomical origin of HSCs also remains a topic of discussion. Many investigators still consider the aortagonad-mesonephros region (AGM) as the sole or main site of HSC generation, although several studies have provided evidence that the placenta and the yolk sac also participate in HSC development (Chen et al., 2009; Gekas et al., 2005; Ottersbach and Dzierzak, 2005; Rhodes et al., 2008). In this issue of Developmental Cell, Pereira et al. (2016) take advantage of their discoveries from in vitro lineage reprogramming studies to define an immediate precursor to mouse HSCs and define their anatomical localization in the mouse conceptus (Figure 1). In an earlier seminal work in 2013, Pereira and colleagues described directed lineage reprogramming of mouse embryonic fibroblasts to hematopoietic stem/progenitor cells (HSPCs) by overexpressing four factors highly expressed in long-term reconstituting HSCs: Gata2, Gfi1b, cFos, and Etv6 (Pereira et al., 2013). Using this approach, they generated early hemogenic precur-
sors (HPs) that expressed Sca1, CD34, and Prom1 (herein called PS34 cells) but were devoid of the pan-hematopoietic marker CD45 expression. Sca1 and CD34 are established markers of HSCs during mouse development, and Prom1 (Prominin1/CD133) has been used as a HSPC marker in humans (Wirthlin et al., 2008). In their new study, Pereira et al. (2016) now identify HP populations with similar PS34 surface phenotype in the developing mouse conceptus and confirm that PS34 cells can be retrieved in multiple sites of HSC development, including the placenta, the AGM region, vitelline, and umbilical vessels (Figure 1). Importantly, the authors show that placental PS34 cells are fetal rather than maternal in origin. This study thus represents a major advance in pinpointing the anatomical origin of definitive HSCs, as it confirms that the placenta is not only a nursery for HSCs during midgestation but also a site of HSC emergence. Future studies will be required to assess whether the yolk sac, another disputed site of HSC development, harbors PS34 HSC precursors. Molecular profiling of placental PS34 cells both at the population and singlecell levels confirmed several expected characteristics of HSC precursors. These include endothelial identity with molecular features of arterial endothelium, as well as the expression of the early endothelial-tohematopoietic transition factors, but the absence of genes expressed in more mature hematopoietic cells. Interestingly, PS34 cells from the placenta clustered together with hemogenic cells obtained by lineage reprogramming at 20 days. Moreover, placental PS34 cells expressed
Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc. 479
Developmental Cell
Previews Etv6 cFos Gata2
Gfi1b
Prom1+Sca1+CD34+ (PS34) cells
fibroblasts
AFT024 co-culture
Placenta PS34
OP9-Dll1 co-culture
HSPC phenotype Colony forming potential
Re-aggregate Placenta co-culture
HSC maturation
BM engraftment
E10.5 Prom1 YFP Lineage tracing AGM
BM seeding
E10.5-12.5 mouse embryo
Figure 1. Lineage Reprogramming of Fibroblasts to Hematopoietic Cells Uncovers the Identity for Hemogenic Precursors that Generates Definitive HSCs in the Placenta and the AGM Top: Enforced expression of four transcription factors (Gata2, Etv6, Gfi1b, and cFos) in mouse embryonic fibroblasts followed by conditioning on hematopoiesis-supportive stroma (AFT024) drives the generation of hemogenic precursors (HPs) with Prom1+Sca1+CD34+ (PS34) surface phenotype. PS34 cells possess the potential to generate HSPCs that produce hematopoietic colonies upon downregulation of the reprogramming factors. Middle: Cells with PS34 phenotype were identified in E10.5–E12.5 mouse conceptus in the placenta vascular labyrinth and in the aorta-gonad-mesonephros region and adjacent arteries. PS34 cells were able to undergo maturation to HSCs in a Notch1-stimulating stroma (OP9-Dll1) and engraft in irradiated mouse recipients. Bottom: Lineage tracing using Prom1-dependent Cre activation at E10.5 labels Prom1-expressing cells with YFP (yellow). These cells are traced into the adult bone marrow (BM) HSCs and their progeny, confirming that Prom1 expression marks definitive HSC precursors.
high levels of Gata2, cFos, and Etv6, the key factors used in hematopoietic lineage reprogramming, and also upregulated the fourth reprogramming factor, Gfi1b, as they transitioned to HSPCs. The authors used state-of-the-art functional assays to show that although PS34 HPs isolated directly from the placenta did not have clonogenic potential, they matured into transplantable HSCs when co-cultured on OP9-Dll1 stroma that induced Notch1 signaling. Secondary transplantation confirmed that these cells were bona fide HSCs. The discovery that PS34 HPs rely on a Notch1-dependent maturation step in culture to acquire engraftment ability is an important advance. While Notch1 is known to be necessary in hemogenic endothelium for proper HSC development (Butko et al.,
2016; Kumano et al., 2003), this study shows that Notch1 signaling is also sufficient to convert early hemogenic precursors to transplantable HSCs in culture. This finding may help improve in vitro protocols for successful HSC generation. Finally, lineage tracing of cells that expressed Prom1 around E10.5 of mouse development verified their contribution to the adult hematopoietic system even in the absence of additional in vitro steps. This study thus provides an incredibly thorough characterization of the immediate HSC precursor at the phenotypic, molecular, and functional levels and offers new approaches to study how HSC precursors mature into functional HSCs. The discovery that PS34 cells generated in vitro through lineage reprogramming have a very similar in vivo counter-
480 Developmental Cell 36, March 7, 2016 ª2016 Elsevier Inc.
part will lead to interesting future studies comparing the similarities and differences between in-vitro-reprogrammed and invivo-derived PS34 cells, which may help identify the missing factors required to obtain engraftable HSCs from lineage-reprogrammed PS34 cells. Importantly, this study shows that the lessons learned from transcription factor-mediated lineage reprogramming are not only useful for engineering cells for regenerative medicine but can also be informative on developmental processes that occur during embryogenesis, which would be otherwise difficult to capture due to the dynamic nature and complexity of these systems. The ability to identify the hemogenic precursors that are on track to become self-renewing HSCs, either in vivo or in vitro, is a major advance in the field and paves the way for establishing protocols to generate HSCs for transplantation therapies.
REFERENCES Butko, E., Pouget, C., and Traver, D. (2016). Dev. Biol. 409, 129–138. Chen, M.J., Yokomizo, T., Zeigler, B.M., Dzierzak, E., and Speck, N.A. (2009). Nature 457, 887–891. Gekas, C., Dieterlen-Lie`vre, F., Orkin, S.H., and Mikkola, H.K. (2005). Dev. Cell 8, 365–375. Jopling, C., Boue, S., and Izpisua Belmonte, J.C. (2011). Nat. Rev. Mol. Cell Biol. 12, 79–89. Kumano, K., Chiba, S., Kunisato, A., Sata, M., Saito, T., Nakagami-Yamaguchi, E., Yamaguchi, T., Masuda, S., Shimizu, K., Takahashi, T., et al. (2003). Immunity 18, 699–711. Ottersbach, K., and Dzierzak, E. (2005). Dev. Cell 8, 377–387. Pereira, C.F., Chang, B., Qiu, J., Niu, X., Papatsenko, D., Hendry, C.E., Clark, N.R., Nomura-Kitabayashi, A., Kovacic, J.C., Ma’ayan, A., et al. (2013). Cell Stem Cell 13, 205–218. Pereira, C.-F., Chang, B., Gomes, A., Bernitz, J., Papatsenko, D., Niu, X., Swiers, G., Azzoni, E., de Bruijn, M.F.T.R., Schaniel, C., et al. (2016). Dev. Cell 36, this issue, 525–539. Rhodes, K.E., Gekas, C., Wang, Y., Lux, C.T., Francis, C.S., Chan, D.N., Conway, S., Orkin, S.H., Yoder, M.C., and Mikkola, H.K. (2008). Cell Stem Cell 2, 252–263. Wirthlin, L., Hess, D., Zhou, P., and Nolta, J. (2008). Biol. Blood Marrow Transplant. 14, 151–153.