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Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies. [ABSTRACT FROM AUTHOR]

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility. [ABSTRACT FROM AUTHOR]

In vitro-generated pluripotent stem cell (PSC)-derived Pax3-induced (iPax3) myogenic progenitors display an embryonic transcriptional signature, but upon engraftment, the profile of re-isolated iPax3 donor-derived satellite cells changes toward similarity with postnatal satellite cells, suggesting that engrafted PSC-derived myogenic cells remodel their transcriptional signature upon interaction within the adult muscle environment. Here, we show that engrafted myogenic progenitors also remodel their metabolic state. Assessment of oxygen consumption revealed that exposure to the adult muscle environment promotes overt changes in mitochondrial bioenergetics, as shown by the substantial suppression of energy requirements in re-isolated iPax3 donor-derived satellite cells compared to their in vitro-generated progenitors. Mass spectrometry-based metabolomic profiling further confirmed the relationship of engrafted iPax3 donor-derived cells to adult satellite cells. The fact that in vitro-generated myogenic progenitors remodel their bioenergetic signature upon in vivo exposure to the adult muscle environment may have important implications for therapeutic applications. [ABSTRACT FROM AUTHOR]

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied. [ABSTRACT FROM AUTHOR]

This commentary provides a brief overview of the steps necessary for the generation of an induced pluripotent stem (iPS) cell‐derived clinical grade product. This process requires extensive, proper documentation as well as a thoughtful and systematic optimization of the manufacturing methods to ensure maintenance of the key biological features of the product, compliance with current good manufacturing practices (cGMP), and most importantly patient safety. The scale‐up and optimization also ideally include the identification of efficient and cost‐effective purification/isolation and expansion of the target cell population. [ABSTRACT FROM AUTHOR]

This article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies. [ABSTRACT FROM AUTHOR]

The transcriptional mechanisms driving lineage specification during development are still largely unknown, as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors—mesogenin 1 (Msgn1), T-box 6 (Tbx6), forkhead box C1 (Foxc1), paired box 3 (Pax3), Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), and myogenic factor 5 (Myf5)—in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null embryonic day (E)9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and bone morphogenetic protein (BMP) signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) factors. To our knowledge, these data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis. [ABSTRACT FROM AUTHOR]

Facioscapulohumeral muscular dystrophy (FSHD) is caused by inappropriate expression of the double homeodomain protein DUX4. DUX4 has bimodal effects, inhibiting myogenic differentiation and blocking MyoD at low levels of expression, and killing myoblasts at high levels. Pax3 and Pax7, which contain related homeodomains, antagonize the cell death phenotype of DUX4 in C2C12 cells, suggesting some type of competitive interaction. Here, we show that the effects of DUX4 on differentiation andMyoD expression require the homeodomains but do not require the C-terminal activation domain of DUX4. We tested the set of equally related homeodomain proteins (Pax6, Pitx2c, OTX1, Rax, Hesx1, MIXL1 and Tbx1) and found that only Pax3 and Pax7 display phenotypic competition. Domain analysis on Pax3 revealed that the Pax3 homeodomain is necessary for phenotypic competition, but is not sufficient, as competition also requires the paired and transcriptional activation domains of Pax3. Remarkably, substitution mutants in which DUX4 homeodomains are replaced by Pax7 homeodomains retain the ability to inhibit differentiation and to induce cytotoxicity. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy (DMD) is an inherited lethal muscle wasting disease characterized by cycles of degeneration and regeneration, with no effective therapy. Growth differentiation factor 11 (GDF11), a member of the TGF-β superfamily and myostatin homologous, has been reported to have the capacity to reverse age-related skeletal muscle loss. These initial findings led us to investigate the ability of GDF11 to promote regeneration in the context of muscular dystrophy and determine whether it could be a candidate to slow down or reverse the disease progression in DMD. Results: Here, we delivered recombinant GDF11 (rGDF11) to dystrophin-deficient mice using the intra-peritoneal route for 30 days and evaluated histology and function in both steady-state and cardiotoxin-injured muscles. Our data confirmed that treatment with rGDF11 resulted in elevated levels of this factor in the circulation. However, this had no effect on muscle contractility nor on muscle histology. Moreover, no difference was found in the number of regenerating myofibers displaying centrally located nuclei. On the other hand, we did observe increased collagen content, which denotes fibrosis, in the muscles of rGDF11-treated dystrophic mice. Conclusions: Taken together, our findings indicate no beneficial effect of treating dystrophic mice with rGDF11 and raise caution to a potential harmful effect, as shown by the pro-fibrotic outcome. [ABSTRACT FROM AUTHOR]

Background: Satellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as selfrenewal and long-term regenerative potential. Methods: We develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice. Results: Here, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human µDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury. Conclusion: These results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) represent an ideal model to study how lineage decisions are established during embryonic development. Using a doxycycline-inducible mouse ESC line, we have previously shown that expression of the transcriptional activator Pax3 in early mesodermal cells leads to the robust generation of paraxial mesoderm progenitors that ultimately differentiate into skeletal muscle precursors. Here, we show that the ability of this transcription factor to induce the skeletal myogenic cell fate occurs at the expenses of the cardiac lineage. Our results show that the PDGFRα+FLK1− subfraction represents the main population affected by Pax3, through downregulation of several transcripts encoding for proteins involved in cardiac development. We demonstrate that although Nkx2-5, Tbx5, and Gata4 negatively affect Pax3 skeletal myogenic activity, the cardiac potential of embryoid body-derived cultures is restored solely by forced expression of Tbx5. Taking advantage of this model, we used an unbiased genome-wide approach to identify genes whose expression is rescued by Tbx5, and which could represent important regulators of cardiac development. These findings elucidate mechanisms regulating the commitment of mesodermal cells in the early embryo and identify the Tbx5 cardiac transcriptome. S tem C ells 2014;32:2072-2083 [ABSTRACT FROM AUTHOR]

ABSTRACT Endoglin (Eng), an ancillary receptor of the transforming growth factor beta (TGF β) signaling pathway superfamily, has been well recognized for its important function in vascular development and angiogenesis since its discovery more than a decade ago. Recent studies show that this receptor is also critical for the emergence of blood during embryonic development, and that at E7.5, endoglin together with Flk-1 identifies early mesoderm progenitors that are endowed with hematopoietic and endothelial potential. These two lineages emerge in very close association during embryogenesis, and because they share the expression of the same surface markers, it has been difficult to distinguish the earliest hematopoietic from endothelial cells. Here, we evaluated the function of endoglin in hematopoiesis as development progresses past E7.5, and found that the hematopoietic and endothelial progenitors can be distinguished by the levels of endoglin in E9.5 yolk sacs. Whereas endothelial cells are Engbright, hematopoietic activity is primarily restricted to a subset of cells that display dim expression of endoglin (Engdim). Molecular characterization of these subfractions showed that endoglin-mediated induction of hematopoiesis occurs in concert with BMP2/BMP4 signaling. This pathway is highly active in Engdim cells but significantly downregulated in the Eng knockout. Taken together, our findings show an important function for endoglin in mediating BMP2/BMP4 signaling during yolk sac hematopoietic development and suggest that the levels of this receptor modulate TGF β versus bone morphogenetic protein (BMP) signaling. S tem C ells 2013;31:1893-1901 [ABSTRACT FROM AUTHOR]

The paired box transcription factor Pax3 is well-known as a major regulator of embryonic myogenesis. Before Pax3 expression becomes restricted to the dermomyotome, this transcription factor is also expressed in the developing somites. The role of Pax3 at this early stage is unclear, in particular because of the scarce frequency of Pax3-positive cells in the early mouse embryo. Inducible gene expression in embryonic stem cells (ESCs) represents an excellent tool to overcome this limitation, since it can provide large quantities of otherwise rare embryonic populations expressing a factor of interest. Here we used engineered mouse ESCs to perform a functional analysis of Pax3 with the aim to identify the molecular determinants involved in the early functions of this transcription factor. We find that Pax3 induction during embryoid body differentiation results in the upregulation of genes expressed in the presomitic and somitic mesoderm. Moreover, we show that paraxial mesoderm induced by transient expression of Pax3 is not irreversibly committed to myogenesis rather requires sustained Pax3 expression. Using a series of deletion mutants of Pax3, which differentially affect its transcriptional activity, we map protein domains necessary for induction of paraxial mesoderm and induction of the myogenic program. The paired, homeo-, and transcriptional activation domains were each required for both processes, however, the paired-c-terminal RED domain showed a paraxial mesoderm-specific activity that was dispensable for myogenesis. These findings demonstrate and provide mechanistic insight into an early role for Pax3 in the generation of paraxial mesoderm. S TEM C ells 2013;31:59-70 [ABSTRACT FROM AUTHOR]

During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 ( Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2+, c-kit+, and CD41+ hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP+ population. In the Etv2 mutant yolk sac, Tie2+ cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2. S TEM C ELLS 2012;30:1611-1623 [ABSTRACT FROM AUTHOR]

An effective long-term cell therapy for skeletal muscle regeneration requires donor contribution to both muscle fibers and the muscle stem cell pool. Although satellite cells have these abilities, their therapeutic potential so far has been limited due to their scarcity in adult muscle. Myogenic progenitors obtained from Pax3-engineered mouse embryonic stem (ES) cells have the ability to generate myofibers and to improve the contractility of transplanted muscles in vivo, however, whether these cells contribute to the muscle stem cell pool and are able to self-renew in vivo are still unknown. Here, we addressed this question by investigating the ability of Pax3, which plays a critical role in embryonic muscle formation, and Pax7, which is important for maintenance of the muscle satellite cell pool, to promote the derivation of self-renewing functional myogenic progenitors from ES cells. We show that Pax7, like Pax3, can drive the expansion of an ES-derived myogenic progenitor with significant muscle regenerative potential. We further demonstrate that a fraction of transplanted cells remains mononuclear, and displays key features of skeletal muscle stem cells, including satellite cell localization, response to reinjury, and contribution to muscle regeneration in secondary transplantation assays. The ability to engraft, self-renew, and respond to injury provide foundation for the future therapeutic application of ES-derived myogenic progenitors in muscle disorders. S C 2011;29:777-790 [ABSTRACT FROM AUTHOR]

Muscle regeneration occurs through activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to make new myofibers. We used a transgenic Pax7- ZsGreen reporter mouse to prospectively isolate stem cells of skeletal muscle by flow cytometry. We show that Pax7- expressing cells (satellite cells) in the limb, head, and diaphragm muscles are homogeneous in size and granularity and uniformly labeled by certain cell surface markers, including CD34 and CD29. The frequency of the satellite cells varies between muscle types and with age. Clonal analysis demonstrated that all colonies arising from single cells within the Pax7-sorted fraction have myogenic potential. In response to injury, Pax7+ cells reduce CD34, CD29, and CXCR4 expression, increase in size, and acquire Sca-1. When directly isolated and cultured in vitro, Pax7+ cells display the hallmarks of activation and proliferate, initially as suspension aggregates and later distributed between suspension and adherence. During in vitro expansion, Pax7 (ZsGreen) and CD34 expression decline, whereas expression of PSA-NCAM is acquired. The nonmyogenic, Pax7neg cells expand as Sca1+ PDGRα+ PSA-NCAMneg cells. Satellite cells expanded exclusively in suspension can engraft and produce dystrophin+ fibers in mdx-/- mice. These results establish a novel animal model for the study of muscle stem cell physiology and a culture system for expansion of engraftable muscle progenitors. [ABSTRACT FROM AUTHOR]

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-α receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy. [ABSTRACT FROM AUTHOR]

Three‐dimensional (3D) skeletal muscle constructs engineered from myogenic progenitors derived from human pluripotent stem cells (hPSCs) have a wide range of applications, but to date, such constructs generate lower specific tetanic force than adult human muscles. Methods enhancing functional muscle differentiation and force generation of these constructs are highly desirable. The finding of this study is that addition of the supplements in the endothelial cell growth medium‐2 (EGM‐2) to the myogenic differentiation medium can substantially enhance contractile force generation. For constructs differentiated for 4 weeks, addition of the EGM‐2 supplements in the first 2 weeks leads to tenfold and sevenfold increases in twitch and tetanic forces, respectively. The specific tetanic force generated by these constructs is 33 mN mm−2, which is significantly higher than previously reported. These constructs show wider myotubes and higher gene expression levels for all skeletal muscle‐specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells underlies the greater contractile force generation. The constructs exposed to these supplements for 4 weeks do not generate as high contractile forces, suggesting that prolonged treatment is not beneficial. These results suggest that temporal conditioning with the EGM‐2 supplements assists functional development of hPSC‐derived skeletal muscle constructs. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is a progressive and incurable neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic induced pluripotent stem cells generated from fibroblasts of mice lacking both dystrophin and utrophin. We correct the phenotype of dystrophic induced pluripotent stem cells using a Sleeping Beauty transposon system carrying the micro-utrophin gene, differentiate these cells into skeletal muscle progenitors and transplant them back into dystrophic mice. Engrafted muscles displayed large numbers of micro-utrophin-positive myofibers, with biochemically restored dystrophin-glycoprotein complex and improved contractile strength. The transplanted cells seed the satellite cell compartment, responded properly to injury and exhibit neuromuscular synapses. We also detect muscle engraftment after systemic delivery of these corrected progenitors. These results represent an important advance towards the future treatment of muscular dystrophies using genetically corrected autologous induced pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Endoglin (ENG), an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, has a well-studied role in endothelial function. Here, we report that endoglin also plays an important role early in development at the level of the hemangioblast, an embryonic progenitor of the hematopoietic and endothelial lineages. Eng-/-, Eng+/- and Eng+/+ mouse embryonic stem (ES) cells were differentiated as embryoid bodies (EBs) and assayed for blast colony-forming cells (BL-CFCs). Our results showed a profound reduction in hemangioblast frequency in the absence of endoglin. Furthermore, cell-sorting experiments revealed that endoglin marks the hemangioblast on day 3 of EB differentiation. When analyzed for hematopoietic and endothelial activity, replated Eng-/- BL-CFCs presented limited hematopoietic potential, whereas endothelial differentiation was unaltered. Analysis of hematopoietic colony formation of EBs, at different time points, further supports a function for endoglin in early hematopoiesis. Taken together, these findings point to a role for endoglin in both hemangioblast specification and hematopoietic commitment. [ABSTRACT FROM AUTHOR]

Endoglin (CD105), a receptor of the transforming growth factor-b superfamily, has been reported to identify functional long-term repopulating hematopoietic stem cells, and has been detected in certain subtypes of acute leukemias. Whether this receptor plays a functional role in leukemogenesis remains unknown. We identified endoglin expression on the majority of blasts from patients with acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Using a xenograft model, we find that CD1051 blasts are endowed with superior leukemogenic activity compared with the CD1052 population. Wetest the effect of targeting this receptor using themonoclonal antibody TRC105, and find that in AML, TRC105 prevented the engraftment of primary AML blasts and inhibited leukemiaprogression following disease establishment, but inB-ALL,TRC105 alonewas ineffective due to the shedding of soluble CD105. However, in both B-ALL and AML, TRC105 synergized with reduced intensity myeloablation to inhibit leukemogenesis, indicating that TRC105 mayrepresent anovel therapeutic optionforB-ALL and AML. [ABSTRACT FROM AUTHOR]

Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here, we show that endoglin (Eng), a receptor for the TGFβ superfamiiy, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng+Flk1+ mesodermal precursor population at embryonic day 7.5 (E7.5), a cell fraction also endowed with endothelial potential. In Eng-knockout embryos, hematopoietic colony activity and numbers of CD71+ Ter119+ erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key bone morphogenic protein (BMP) target genes, including the hematopoietic regulators Sel, Gâtai, Gata2, and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng+Flk-1+ progenitors coex-pressed TGFβ and BMP receptors and target genes. Furthermore, Eng+Flk-1+ cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of Eng-expressing cells was dependent on the TGFβ superfamiiy ligands BMP4, BMP2, or TGF-β1. These data demonstrate that the E+F+ fraction at E7.5 represents mesodermal cells competent to respond to TGFβl, BMP4, or BMP2, shaping their hematopoietic development, and that Eng acts as a critical regulator in this process by modulating TGF/BMP signaling. [ABSTRACT FROM AUTHOR]

Endoglin (Eng), an accessory receptor for the transforming growth factor β (TGF-β) superfamily, Is required for proper hemangioblast and primitive hematopoietic development. However the mechanism by which endoglin functions at this early developmental stage is currently unknown. Transcriptional analyses of differentiating eng-/- and engng-/- ES cells revealed that lack of endogiin leads to profound reductions in the levels of key hematopoietlc regulators, including Sd, Lmo2, and Gata2. We also detected lower levels of phosphorylated Smadi (pSmadi), a downstream target signaling moiecuie associated with the TGF-β pathway. Using doxycycline-inducible ES cell lines, we interrogated the TGF-β signaling pathway by expressing activated forms of ALK-1 and ALK-5, type i receptors for TGF-β. Our results indicate that ALK-1 signaling promotes hemangloblast development and hematopoiesis, as evidenced by colony assays, gene expression and FACS anaiyses, whereas signaling by ALK-5 leads to the opposite effect, inhibition of hemangioblast and hematopoietic development. In Eng-/- ES cells, ALK-1 rescued both the defective hemangioblast development, and primitive erythropoiesis, indicating that ALK-1 signaling can compensate for the absence of endoglin. We propose that endoglin regulates primitive hematopolesis by modulating the activity of the Smadl/5 signaling pathway in early stages of development. [ABSTRACT FROM AUTHOR]