Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.     

BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.     

Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling

The canonical Wnt/beta-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of beta-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of beta-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing beta-catenin stability. In addition, Activin/Nodal and Wnt/beta-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/beta-catenin signaling in hES cells.     

A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.     

A high-throughput multiplexed screening assay for optimizing serum-free differentiation protocols of human embryonic stem cells

Serum-free differentiation protocols of human embryonic stem cells (hESCs) offer the ability to maximize reproducibility and to develop clinically applicable therapies. We developed a high-throughput, 96-well plate, four-color flow cytometry-based assay to optimize differentiation media cocktails and to screen a variety of conditions. We were able to differentiate hESCs to all three primary germ layers, screen for the effect of a range of activin A, BMP4, and VEGF concentrations on endoderm and mesoderm differentiation, and perform RNA-interference (RNAi)-mediated knockdown of a reporter gene during differentiation. Cells were seeded in suspension culture and embryoid bodies were induced to differentiate to the three primary germ layers for 6 days. Endoderm (CXCR4(+)KDR(-)), mesoderm (KDR(+)SSEA-3(-)), and ectoderm (SSEA-3(+)NCAM(+)) differentiation yields for H9 cells were 80 ± 11, 78 ± 7, and 41 ± 9%, respectively. Germ layer identities were confirmed by quantitative PCR. Activin A, BMP4, and bFGF drove differentiation, with increasing concentrations of activin A inducing higher endoderm yields and increasing BMP4 inducing higher mesoderm yields. VEGF drove lateral mesoderm differentiation. RNAi-mediated knockdown of constitutively expressed red fluorescent protein did not affect endoderm differentiation. This assay facilitates the development of serum-free protocols for hESC differentiation to target lineages and creates a platform for screening small molecules or RNAi during ESC differentiation.     

Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood

The embryonic stem cell differentiation system was used to define the roles of the Activin/Nodal, BMP, and canonical Wnt signaling pathways at three distinct developmental stages during hematopoietic ontogeny: induction of a primitive streak-like population, formation of Flk1(+) mesoderm, and induction of hematopoietic progenitors. Activin/Nodal and Wnt, but not BMP, signaling are required for the induction of the primitive streak. Although BMP is not required for primitive streak induction, it displays a strong posteriorizing effect on this population. All three signaling pathways regulate induction of Flk1(+) mesoderm. The specification of Flk1(+) mesoderm to the hematopoietic lineages requires VEGF and Wnt, but not BMP or Activin/Nodal signaling. Specifically, Wnt signaling is essential for commitment of the primitive erythroid, but not the definitive lineages. These findings highlight dynamic changes in signaling requirements during blood cell development and identify a role for Wnt signaling in the establishment of the primitive erythroid lineage.     

Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines

Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression, we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly, we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts.     

人多能干细胞来源的肾定向分化平台的构建

肾是一种重要的人体器官,具有多种生理功能。然而,全球范围内约有10%的人口患有肾疾病。因此,建立一种接近人体肾的结构与功能的模型进行肾疾病的研究是十分必要的。多能干细胞体外定向诱导分化技术的兴起,为再生医学和精准医学领域注入了新的动力。本研究通过在体外条件下模拟体内肾发育的过程,将人多能干细胞包括胚胎干细胞和诱导多能干细胞,通过体外定向诱导分化形成肾的祖细胞,进而建立肾的结构与功能单位:肾元。该研究通过激活WNT信号通路,同时抑制TGF-β信号通路,将人多能干细胞从多能态定向诱导至原条阶段。之后通过细胞自分化的能力使其发育至中间中胚层,再通过激活FGF信号通路,将其分化至肾祖细胞阶段。流式细胞检测结果显示,肾祖细胞占总细胞数的51.5%~61.9%。通过免疫荧光检测发现:分化得到的结构中包含肾小球足细胞、近端小管、远端小管等肾组织结构。该研究建立的肾体外分化方法,具有稳定性好、分化效率高、重复性好的特点。为研究人类肾的早期发育机制,肾疾病模型构建,以及药物筛选提供了一种新的方法。     

Defining early hematopoietic-fated primitive streak specification of human pluripotent stem cells by the orchestrated balance of Wnt,activin, and BMP signaling

Distinct regions of the primitive streak (PS) have diverse potential to differentiate into several tissues, including the hematopoietic lineage originated from the posterior region of PS. Although various signaling pathways have been identified to promote the development of PS and its mesoderm derivatives, there is a large gap in our understanding of signaling pathways that regulate the hematopoietic fate of PS. Here, we defined the roles of Wnt, activin, and bone morphogenetic protein (BMP) signaling pathways in generating hematopoietic-fated PS from human pluripotent stem cells (hPSCs). We found that the synergistic balance of these signaling pathways was crucial for controlling the PS fate determination towards hematopoietic lineage via mesodermal progenitors. Although the induction of PS depends largely on the Wnt and activin signaling, the PS generated without BMP4 lacks the hematopoietic potential, indicating that the BMP signaling is necessary for the PS to acquire hematopoietic property. Appropriate levels of Wnt signaling is crucial for the development of PS and its specification to the hematopoietic lineage. Although the development of PS is less sensitive to activin or BMP signaling, the fate of PS to mesoderm progenitors and subsequent hematopoietic lineage is determined by appropriate levels of activin or BMP signaling. Collectively, our study demonstrates that Wnt, activin, and BMP signaling pathways play cooperative and distinct roles in regulating the fate determination of PS for hematopoietic development. Our understanding of the regulatory networks of hematopoietic-fated PS would provide important insights into early hematopoietic patterning and possible guidance for generating functional hematopoietic cells from hPSCs in vitro.     

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.     

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-α (PDGFR-α) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-α and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm

Human embryonic stem cells (hESCs) can provide insights into development of inaccessible human tissues such as embryonic endoderm. Progress in this area has been hindered by a lack of methods for isolating endodermal cells and tracing fates of their differentiated progeny. By using homologous recombination in human ESCs, we inserted an enhanced green fluorescent protein (eGFP) transgene into the SOX17 locus, a postulated marker of human endoderm. FACS purification and gene expression profiling confirmed that SOX17(+)-hESC progeny expressed endodermal markers and unveiled specific cell surface protein combinations that permitted FACS-based isolation of primitive gut tube endodermal cells produced from unmodified human ESCs and from induced pluripotent stem cells (iPSC). Differentiating SOX17(+) endodermal cells expressed markers of liver, pancreas, and intestinal epithelium in vitro and gave rise to endodermal progeny in vivo. Thus, prospective isolation, lineage tracing, and developmental studies of SOX17(+) hESC progeny have revealed fundamental aspects of human endodermal biology.     

Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Blood islands' of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper. These lineages arise from a layer of extraembryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules (Indian hedgehog) is produced by visceral endoderm, can induce formation of blood and endothelial cells in explant cultures and can reprogram prospective neurectoderm along hematopoietic and endothelial cell lineages. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. These findings may have important implications for regulating hematopoiesis and vascular development for therapeutic purposes in humans and for the development of new sources of stem cells for transplantation and gene therapy.     

ADMP2 is essential for primitive blood and heart development in Xenopus

We describe here the cloning of a new member of the TGF-beta family with similarity to the anti-dorsalizing morphogenetic proteins (ADMPs). This new gene, ADMP2, is expressed in a broad band of mesendoderm cells that appear to include the progenitors of the endoderm and the ventral mesoderm. Antisense morpholino oligonucleotide knockdown of ADMP2 results in near-complete disruption of primitive blood and heart development, while the development of other mesoderm derivatives, including pronephros, muscle and lateral plate is not disrupted. Moreover, the development of the primitive blood in ADMP2 knockdown embryos cannot be rescued by BMP. These results suggests that ADMP2 plays an early role in specifying presumptive ventral mesoderm in the leading edge mesoderm, and that ADMP2 activity may be necessary to respond to BMP signaling in the context of ventral mesoderm induction.     

Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning

Anteroposterior (AP) polarity in the mammalian embryo is specified during gastrulation when naive progenitor cells in the primitive ectoderm are recruited into the primitive streak to form mesoderm and endoderm. At the opposite pole, this process is inhibited by signals previously induced in distal visceral endoderm (DVE). Both DVE and primitive streak formation, and hence positioning of the AP axis, rely on the TGFbeta family member Nodal and its proprotein convertases Furin and Pace4. Here, we show that Nodal and Furin are initially co-expressed in the primitive endoderm together with a subset of DVE markers such as Lefty1 and Hex. However, with the appearance of extra-embryonic ectoderm (ExE), DVE formation is transiently inhibited. During this stage, Nodal activity is essential to specify embryonic VE and restrict the expression of Furin to the extra-embryonic region. Activation of Nodal is also necessary to maintain determinants of pluripotency such as Oct4, Nanog and Foxd3 during implantation, and to stimulate elongation of the egg cylinder, before inducing DVE and germ layer formation. We conclude that Nodal is already activated in primitive endoderm, but induces a functional DVE only after promoting the expansion of embryonic VE and pluripotent progenitor cells in the epiblast.     

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.