Organoids derived from human pluripotent stem cells (hPSCs) have emerged as important models for investigating human-specific aspects of development and disease. Here we discuss hPSC-derived ...organoids through the lens of development—highlighting how stages of human development align with the development of hPSC-derived organoids in the tissue culture dish. Using hPSC-derived lung and intestinal organoids as examples, we discuss the value and application of such systems for understanding human biology, as well as strategies for enhancing organoid complexity and maturity.
Highlights • The first two cell fate decisions in mammalian development create ES cell progenitors and two extraembryonic cell types that are essential for fetal development. • During blastocyst ...formation, cell fates are initially specified by cell signaling, and are then reinforced by lineage-specific transcription factors. • Technological advances in imaging and gene expression analysis enable new insights into the mechanisms of cell fate specification in the mouse early embryo.
During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway ...in controlling cell positioning and expression of
, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress
while promoting expression of the trophectoderm gene
in parallel. Yet,
is more sensitive than
to
dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating
expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.
The article hPSC-derived organoids: models of human development and disease, written by Tristan Frum and Jason R. Spence, was originally published electronically on the publisher’s internet portal on ...28 August 2020 without open access.
In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage.
is the only pluripotency gene known to be expressed specifically within inside cells at ...this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of
expression. Surprisingly,
expression initiated normally in the absence of both
and
(
), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of
prior to the 16-cell stage in the absence of
,
and
Interestingly, the repression of premature
expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of
by YAP1, WWTR1 and TEAD4
Taken together, our observations reveal that, while embryos are initially competent to express
as early as the four-cell stage, transcriptional repression prevents the premature expression of
, thereby restricting the pluripotency program to the stage when inside cells are first created.
Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes ...regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and ...translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.
In embryonic stem (ES) cells and in early mouse embryos, the transcription factor Oct4 is an essential regulator of pluripotency. Oct4 transcriptional targets have been described in ES cell lines; ...however, the molecular mechanisms by which Oct4 regulates establishment of pluripotency in the epiblast (EPI) have not been fully elucidated. Here, we show that neither maternal nor zygotic Oct4 is required for the formation of EPI cells in the blastocyst. Rather, Oct4 is first required for development of the primitive endoderm (PE), an extraembryonic lineage. EPI cells promote PE fate in neighboring cells by secreting Fgf4, and Oct4 is required for expression of Fgf4, but we show that Oct4 promotes PE development cell-autonomously, downstream of Fgf4 and Mapk. Finally, we show that Oct4 is required for the expression of multiple EPI and PE genes as well as multiple metabolic pathways essential for the continued growth of the preimplantation embryo.
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•Maternal Oct4 is dispensable for development•Oct4 promotes primitive endoderm and represses trophectoderm fates in parallel•Oct4 acts upstream of Fgf4 in epiblast and downstream of Mapk in primitive endoderm•Oct4 is required for expression of metabolic pathway genes in the blastocyst
In ES cells, Oct4 opposes differentiation and maintains pluripotency. Here, Frum et al. show that in mouse blastocysts, Oct4 plays an opposing role promoting development of primitive endoderm, a differentiated lineage. Zygotic Oct4 enables primitive endoderm cells to respond to neighboring epiblast cells, while maternal Oct4 is dispensable for development.
In many invertebrate and vertebrate species, cell fates are assigned through the cellular inheritance of differentially localized maternal determinants. Whether mammalian embryogenesis is also ...regulated by deterministic mechanisms is highly controversial. The caudal domain transcription factor CDX2 has been reported to act as a maternal determinant regulating cell fate decisions in mouse development. However, this finding is contentious because of reports that maternal Cdx2 is not essential for development. Notably, all of the previously published studies of maternal Cdx2 relied on injected RNA interference constructs, which could introduce experimental variation. Only deletion of the maternal gene can unambiguously resolve its requirement in mouse development. Here, we genetically ablated maternal Cdx2 using a Cre/lox strategy, and we definitively establish that maternal Cdx2 is not essential for mouse development.
The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These “bud tip progenitors” are regulated by reciprocal signaling with surrounding ...mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.
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•scRNA-seq of developing human distal lung mesenchyme identified cellular heterogeneity•RSPO2+ mesenchymal cells lie adjacent to LGR5+ epithelial bud tip progenitors•Blocking RSPO2/LGR5 in vitro reduced WNT signaling and led to airway differentiation•RSPO2+ mesenchyme provides a niche for bud tips in co-cultures
Hein et al. present the scRNA-seq of the developing human distal lung, showing transcriptionally distinct populations of mesenchyme. Spatial profiling showed that RSPO2+ mesenchymal cells are physically adjacent to LGR5+ epithelial bud tips. Organoid experiments revealed that RSPO2+ cells create a high WNT signaling environment, supporting bud tip identity and multipotency.
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