Cripto Is Targeted by miR-1a-3p in a Mouse Model of Heart Development.

During cardiac differentiation, numerous factors contribute to the development of the heart. Understanding the molecular mechanisms underlying cardiac development will help combat cardiovascular disorders, among the leading causes of morbidity and mortality worldwide. Among the main mechanisms, we indeed find Cripto. Cripto is found in both the syncytiotrophoblast of ampullary pregnancies and the inner cell mass along the primitive streak as the second epithelial-mesenchymal transformation event occurs to form the mesoderm and the developing myocardium. At the same time, it is now known that cardiac signaling pathways are intimately intertwined with the expression of myomiRNAs, including miR-1. This miR-1 is one of the muscle-specific miRs; aberrant expression of miR-1 plays an essential role in cardiac diseases. Given this scenario, our study aimed to evaluate the inverse correlation between Cripto and miR-1 during heart development. We used in vitro models of the heart, represented by embryoid bodies (EBs) and embryonic carcinoma cell lines derived from an embryo-derived teratocarcinoma in mice (P19 cells), respectively. First, through a luciferase assay, we demonstrated that Cripto is a target of miR-1. Following this result, we observed that as the days of differentiation increased, the Cripto gene expression decreased, while the level of miR-1 increased; furthermore, after silencing miR-1 in P19 cells, there was an increase in Cripto expression. Moreover, inducing damage with a cobra cardiotoxin (CTX) in post-differentiation cells, we noted a decreased miR-1 expression and increased Cripto. Finally, in mouse cardiac biopsies, we observed by monitoring gene expression the distribution of Cripto and miR-1 in the right and left ventricles. These results allowed us to detect an inverse correlation between miR-1 and Cripto that could represent a new pharmacological target for identifying new therapies.

Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle.

Macrophages are characterized by a high plasticity in response to changes in tissue microenvironment, which allows them to acquire different phenotypes and to exert essential functions in complex processes, such as tissue regeneration. Here, we report that the membrane protein Cripto plays a key role in shaping macrophage plasticity in skeletal muscle during regeneration and disease. Conditional deletion of Cripto in the myeloid lineage (CriptoMy-LOF ) perturbs MP plasticity in acutely injured muscle and in mouse models of Duchenne muscular dystrophy (mdx). Specifically, CriptoMy-LOF macrophages infiltrate the muscle, but fail to properly expand as anti-inflammatory CD206+ macrophages, which is due, at least in part, to aberrant activation of TGFß/Smad signaling. This reduction in macrophage plasticity disturbs vascular remodeling by increasing Endothelial-to-Mesenchymal Transition (EndMT), reduces muscle regenerative potential, and leads to an exacerbation of the dystrophic phenotype. Thus, in muscle-infiltrating macrophages, Cripto is required to promote the expansion of the CD206+ anti-inflammatory macrophage type and to restrict the EndMT process, providing a direct functional link between this macrophage population and endothelial cells.

Long non-coding RNA in stem cell pluripotency and lineage commitment: functions and evolutionary conservation.

LncRNAs have recently emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression. Indeed, lncRNAs participate in a wide variety of stem cell and developmental processes, acting in cis and/or in trans in the nuclear and/or in the cytoplasmic compartments, and generating an intricate network of interactions with RNAs, enhancers, and chromatin-modifier complexes. Given the versatility of these molecules to operate in different subcellular compartments, via different modes of action and with different target specificity, the interest in this research field is rapidly growing. Here, we review recent progress in defining the functional role of lncRNAs in stem cell biology with a specific focus on the underlying mechanisms. We also discuss recent findings on a new family of evolutionary conserved lncRNAs transcribed from ultraconserved elements, which show perfect conservation between human, mouse, and rat genomes, and that are emerging as new player in this complex scenario.

Hhex and Cer1 mediate the Sox17 pathway for cardiac mesoderm formation in embryonic stem cells.

Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm-derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome-wide profiling of Sox17-dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over-expression of Sox17 induced endogenous Cer1 and sequence-specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex-deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2.

Cripto regulates skeletal muscle regeneration and modulates satellite cell determination by antagonizing myostatin.

Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-ß ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.

Cellular interactions and microenvironment dynamics in skeletal muscle regeneration and disease.

Skeletal muscle regeneration relies on the intricate interplay of various cell populations within the muscle niche-an environment crucial for regulating the behavior of muscle stem cells (MuSCs) and ensuring postnatal tissue maintenance and regeneration. This review delves into the dynamic interactions among key players of this process, including MuSCs, macrophages (MPs), fibro-adipogenic progenitors (FAPs), endothelial cells (ECs), and pericytes (PCs), each assuming pivotal roles in orchestrating homeostasis and regeneration. Dysfunctions in these interactions can lead not only to pathological conditions but also exacerbate muscular dystrophies. The exploration of cellular and molecular crosstalk among these populations in both physiological and dystrophic conditions provides insights into the multifaceted communication networks governing muscle regeneration. Furthermore, this review discusses emerging strategies to modulate the muscle-regenerating niche, presenting a comprehensive overview of current understanding and innovative approaches.

Protocol for characterizing non-genetic heterogeneity and expression dynamics of surface proteins in mouse muscle stem cells using flow cytometry.

Here, we present a protocol for investigating the non-genetic heterogeneity of membrane proteins expression within murine muscle stem cell (MuSC) population isolated from injured skeletal muscles. We describe a protocol that employs flow cytometry technology to detect variations in membrane CRIPTO protein levels and ensure measurements standardization. We detail steps for muscle digestion, bulk muscle cell staining, and phenotypic analysis. This approach allows for the identification of MuSC fractions with distinct phenotypic and functional properties. For complete details on the use and execution of this protocol, please refer to Guardiola et al.1.

Development of a local controlled release system for therapeutic proteins in the treatment of skeletal muscle injuries and diseases.

The present study aims to develop and characterize a controlled-release delivery system for protein therapeutics in skeletal muscle regeneration following an acute injury. The therapeutic protein, a membrane-GPI anchored protein called Cripto, was immobilized in an injectable hydrogel delivery vehicle for local administration and sustained release. The hydrogel was made of poly(ethylene glycol)-fibrinogen (PEG-Fibrinogen, PF), in the form of injectable microspheres. The PF microspheres exhibited a spherical morphology with an average diameter of approximately 100 micrometers, and the Cripto protein was uniformly entrapped within them. The release rate of Cripto from the PF microspheres was controlled by tuning the crosslinking density of the hydrogel, which was varied by changing the concentration of poly(ethylene glycol) diacrylate (PEG-DA) crosslinker. In vitro experiments confirmed a sustained-release profile of Cripto from the PF microspheres for up to 27 days. The released Cripto was biologically active and promoted the in vitro proliferation of mouse myoblasts. The therapeutic effect of PF-mediated delivery of Cripto in vivo was tested in a cardiotoxin (CTX)-induced muscle injury model in mice. The Cripto caused an increase in the in vivo expression of the myogenic markers Pax7, the differentiation makers eMHC and Desmin, higher numbers of centro-nucleated myofibers and greater areas of regenerated muscle tissue. Collectively, these results establish the PF microspheres as a potential delivery system for the localized, sustained release of therapeutic proteins toward the accelerated repair of damaged muscle tissue following acute injuries.

Three-dimensional environment sensitizes pancreatic cancer cells to the anti-proliferative effect of budesonide by reprogramming energy metabolism.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower occurrence of PDAC has been described in individuals with severe and long-standing asthma. Here we explored the potential link between PDAC and the glucocorticoid (GC) budesonide, a first-line therapy to treat asthma. METHODS: We tested the effect of budesonide and the classical GCs on the morphology, proliferation, migration and invasiveness of patient-derived PDAC cells and pancreatic cancer cell lines, using 2D and 3D cultures in vitro. Furthermore, a xenograft model was used to investigate the effect of budesonide on PDAC tumor growth in vivo. Finally, we combined genome-wide transcriptome analysis with genetic and pharmacological approaches to explore the mechanisms underlying budesonide activities in the different environmental conditions. RESULTS: We found that in 2D culture settings, high micromolar concentrations of budesonide reduced the mesenchymal invasive/migrating features of PDAC cells, without affecting proliferation or survival. This activity was specific and independent of the Glucocorticoid Receptor (GR). Conversely, in a more physiological 3D environment, low nanomolar concentrations of budesonide strongly reduced PDAC cell proliferation in a GR-dependent manner. Accordingly, we found that budesonide reduced PDAC tumor growth in vivo. Mechanistically, we demonstrated that the 3D environment drives the cells towards a general metabolic reprogramming involving protein, lipid, and energy metabolism (e.g., increased glycolysis dependency). This metabolic change sensitizes PDAC cells to the anti-proliferative effect of budesonide, which instead induces opposite changes (e.g., increased mitochondrial oxidative phosphorylation). Finally, we provide evidence that budesonide inhibits PDAC growth, at least in part, through the tumor suppressor CDKN1C/p57Kip2. CONCLUSIONS: Collectively, our study reveals that the microenvironment influences the susceptibility of PDAC cells to GCs and provides unprecedented evidence for the anti-proliferative activity of budesonide on PDAC cells in 3D conditions, in vitro and in vivo. Our findings may explain, at least in part, the reason for the lower occurrence of pancreatic cancer in asthmatic patients and suggest a potential suitability of budesonide for clinical trials as a therapeutic approach to fight pancreatic cancer.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Budesonide Analogues Preserve Stem Cell Pluripotency and Delay 3D Gastruloid Development.

Small molecules that can modulate or stabilize cell-cell interactions are valuable tools for investigating the impact of collective cell behavior on various biological processes such as development/morphogenesis, tissue regeneration and cancer progression. Recently, we showed that budesonide, a glucocorticoid widely used as an anti-asthmatic drug, is a potent regulator of stem cell pluripotency. Here we tested the effect of different budesonide derivatives and identified CHD-030498 as a more effective analogue of budesonide. CHD-030498 was able to prevent stem cell pluripotency exit in different cell-based models, including embryonic stem-to-mesenchymal transition, spontaneous differentiation and 3D gastruloid development, and at lower doses compared to budesonide.

Cripto recruits Furin and PACE4 and controls Nodal trafficking during proteolytic maturation.

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

Generation of Epiblast-Like Cells.

Different states of pluripotency can be captured in vitro depending on the embryo stage from which they are derived and the culture conditions. Pluripotency is a continuum of different states between the two extremes of naïve embryonic stem cells (ESCs) and primed Epiblast Stem Cells (EpiSCs), which resemble the pre/peri- and post- implantation embryo, respectively. The transition from naïve to primed pluripotency can be induced by growing naïve ESCs in EpiSCs medium, containing bFGF and Activin. Here we report the detailed protocol to generate and characterize the epiblast-like cells (EpiLCs), which correspond to a primed intermediate state between naïve ESCs and EpiSCs.

Generation of Gastruloids from Epiblast-Like Cells.

The different states of mouse pluripotency described so far rely on a combination of molecular, phenotypic, and functional analysis. Embryonic Stem cells (ESCs) aggregated in suspension culture are able to form 3D embryo-like structures called gastruloids that mimic features of the gastrulation process. Recent findings indicate that gastruloid formation efficiency decreases as pluripotency progresses from naïve to primed state, and suggest that gastruloids formation may represent a functional assay to discriminate different states of mouse pluripotency.Here we describe a method to generate gastruloids from Epiblast-like cells (EpiLCs), which are transiently induced from ESCs by Activin A and bFGF and represent an intermediate state from naïve ESCs to primed Epiblast Stem cells.

Capturing Transitional Pluripotency through Proline Metabolism.

In this paper, we summarize the current knowledge of the role of proline metabolism in the control of the identity of Embryonic Stem Cells (ESCs). An imbalance in proline metabolism shifts mouse ESCs toward a stable naïve-to-primed intermediate state of pluripotency. Proline-induced cells (PiCs), also named primitive ectoderm-like cells (EPLs), are phenotypically metastable, a trait linked to a rapid and reversible relocalization of E-cadherin from the plasma membrane to intracellular membrane compartments. The ESC-to-PiC transition relies on the activation of Erk and Tgfß/Activin signaling pathways and is associated with extensive remodeling of the transcriptome, metabolome and epigenome. PiCs maintain several properties of naïve pluripotency (teratoma formation, blastocyst colonization and 3D gastruloid development) and acquire a few traits of primed cells (flat-shaped colony morphology, aerobic glycolysis metabolism and competence for primordial germ cell fate). Overall, the molecular and phenotypic features of PiCs resemble those of an early-primed state of pluripotency, providing a robust model to study the role of metabolic perturbations in pluripotency and cell fate decisions.

CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

Chemotherapy is the mainstay for the treatment of non-small cell lung cancer (NSCLC). However, NSCLC cells are either intrinsically chemoresistant or rapidly develop therapy resistance. Cancer stem cells (CSCs) are widely recognized as the cell population responsible for resistance to systemic therapies, but the molecular responses of CSCs to chemotherapeutic agents are largely unknown. We identified the embryonic protein CRIPTO in stem cell-enriched spheroid cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC) derived from NSCLC surgical specimens. The CRIPTO-positive population had increased clonogenic capacity and expression of stem cell-related factors. Stemness-related properties were also obtained with forced CRIPTO expression, whereas CRIPTO downregulation resulted in cell cycle blockade and CSCs death. Cell populations positive and negative for CRIPTO expression were interconvertible, and interfering with their reciprocal equilibrium resulted in altered homeostasis of cell expansion both in spheroid cultures and in tumor xenografts. Chemotherapy treatment of NSCLC cells resulted in reduction of cell number followed by increased CRIPTO expression and selective survival of CRIPTO-positive cells. In NSCLC tumor xenografts, chemotherapeutic agents induced partial cell death and tumor stabilization followed by CRIPTO overexpression and tumor progression. Altogether, these findings indicate CRIPTO as a marker of lung CSCs possibly implicated in cancer cell plasticity and post-chemotherapy tumor progression.

Stabilization of cell-cell adhesions prevents symmetry breaking and locks in pluripotency in 3D gastruloids.

3D embryonic stem cell (ESC) aggregates self-organize into embryo-like structures named gastruloids that recapitulate the axial organization of post-implantation embryos. Crucial in this process is the symmetry-breaking event that leads to the emergence of asymmetry and spatially ordered structures from homogeneous cell aggregates. Here, we show that budesonide, a glucocorticoid drug widely used to treat asthma, prevents ESC aggregates to break symmetry. Mechanistically, the effect of budesonide is glucocorticoid receptor independent. RNA sequencing and lineage fate analysis reveal that budesonide counteracts exit from pluripotency and modifies the expression of a large set of genes associated with cell migration, A-P axis formation, and WNT signaling. This correlates with reduced phenotypic and molecular cell heterogeneity, persistence of E-CADHERIN at the cell-cell interface, and cell aggregate compaction. Our findings reveal that cell-cell adhesion properties control symmetry breaking and cell fate transition in 3D gastruloids and suggest a potential adverse effect of budesonide on embryo development.

Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation.

During recent years, increased efforts have focused on elucidating the pluripotency and self-renewal of stem cells. Differentiation towards the different lineages has attracted significant attention given the potential use of stem cells in regenerative medicine. Embryonic stem cell differentiation is a complex process coordinated by strictly regulated extracellular signals that act in an autocrine and/or paracrine manner. Through secreted molecules, stem cells affect local niche biology and influence the cross-talking with the surrounding tissues. Emerging evidence supports the hypothesis that fundamental cell functions, including proliferation and differentiation, are strictly regulated by the complex set of molecules secreted from cells. The understanding of this molecular language could largely increase our knowledge on pathways regulating stem cell differentiation. Here, we have used a proteomics platform to investigate the profile of proteins secreted during differentiation of murine embryonic stem cells. We have followed the dynamics of protein secretion by comparing the secretomes at different time points of murine embryonic stem cell cardiac and neural differentiation. In addition to previously reported molecules, we have identified many secreted proteins not described so far as released from embryonic stem cells nor shown to be differentially released during the process of cardiomyogenesis and neurogenesis.

A small synthetic cripto blocking Peptide improves neural induction, dopaminergic differentiation, and functional integration of mouse embryonic stem cells in a rat model of Parkinson's disease.

Cripto is a glycosylphosphatidylinositol-anchored coreceptor that binds Nodal and the activin type I (ALK)-4 receptor, and is involved in cardiac differentiation of mouse embryonic stem cells (mESCs). Interestingly, genetic ablation of cripto results in increased neuralization and midbrain dopaminergic (DA) differentiation of mESCs, as well as improved DA cell replacement therapy (CRT) in a model of Parkinson's disease (PD). In this study, we developed a Cripto specific blocking tool that would mimic the deletion of cripto, but could be easily applied to embryonic stem cell (ESC) lines without the need of genetic manipulation. We thus screened a combinatorial peptide library and identified a tetrameric tripeptide, Cripto blocking peptide (BP), which prevents Cripto/ALK-4 receptor interaction and interferes with Cripto signaling. Cripto BP treatment favored neuroectoderm formation and promoted midbrain DA neuron differentiation of mESCs in vitro and in vivo. Remarkably, Cripto BP-treated ESCs, when transplanted into the striatum of PD rats, enhanced functional recovery and reduced tumor formation, mimicking the effect of genetic ablation of cripto. We therefore suggest that specific blockers such as Cripto BP may be used to improve the differentiation of ESC-derived DA neurons in vitro and their engraftment in vivo, bringing us closer towards an application of ESCs in CRT.