Mesodermal Differentiation Research Articles

Gastruloids are competent to specify both cardiac and skeletal muscle lineages

Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Here, we reproduce cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages with gastruloids from mouse embryonic stem cells. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm development and differentiation in gastruloids compared to mouse embryos, we present the evidence for skeletal myogenesis in gastruloids. We identify different subpopulations of cardiomyocytes and skeletal muscles, the latter of which most likely correspond to different states of myogenesis with “head-like” and “trunk-like” skeletal myoblasts. In this work, we unveil the potential of gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases.

Prenatal glucocorticoid exposure and congenital abdominal wall defects: Involvement of CXCR4 – SDF-1 signaling

Developmental defects of the ventral abdominal wall, such as gastroschisis, have been associated with prenatal stress exposure. To investigate this further, dexamethasone (DEX), a synthetic glucocorticoid, was administered to fertilized chicken eggs on day 1 of incubation to simulate stress, and embryonic development was subsequently analyzed through in-situ hybridization, immunohistochemistry, and histological methods. Significant developmental abnormalities were displayed by DEX-treated embryos, including open abdomens, reduced MYOG expression in the abdominal wall, and disrupted muscle fiber formation, as indicated by altered Myosin heavy chain patterns. Additionally, early markers of muscle development, such as Pax3, and the CXCR4-SDF-1 signaling axis, crucial for the migration of myogenic precursors of the dermomyotome, were markedly affected. Significant alterations in the expression of mesenchymal markers, including Vimentin and Fibronectin in the lateral plate mesoderm, were observed, alongside alterations in Pitx2, BMP4 and TFAP2A expression. Importantly, a downregulation of Glucocorticoid Receptors was identified, emphasizing the chronic stress exposure. These results provide critical insights into how DEX interferes with key developmental pathways, particularly those involving chemokines like CXCR4 and SDF-1, and other markers of mesodermal differentiation. An advancement in the understanding of the mechanisms underlying ventral abdominal wall defects in the context of prenatal stress is provided by this research, with potential implications for preventing these congenital anomalies.

The Glu86 Residue in TBX4 Proves Critical for Human Lung Development.

T-box transcription factors are a group of evolutionarily conserved T-box-containing regulators of mesoderm specification and development. Heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions involving dosage-sensitive TBX4 have been associated with pulmonary arterial hypertension (PAH), ischiocoxopodopatellar syndrome with or without PAH, and lethal lung developmental disorders (LLDDs), including acinar dysplasia (AcDys), congenital alveolar dysplasia (CAD), and other unspecified primary pulmonary hypoplasias. Loss- and gain-of-function variants have been proposed to cause pediatric PAH and LLDDs, and adult forms of PAH, respectively. Of more than 50 missense SNVs scattered across the entire TBX4, only three have been reported in patients with LLDDs, all mapping to the T-box domain. Here, we report a recurrence of a pathogenic substitution Glu86Lys identified in an unrelated patient with AcDys. In silico predictions of the conformational changes of TBX4 resulting from this and another substitution, Glu86Gln, suggest the loss of most intermolecular hydrogen bonds involving residue 86, including those with Tyr230 that directly interact with DNA. Functional assays on the TBX4 variants in fetal lung fibroblasts confirmed their deleterious character. We propose that Glu86 is critically involved in maintaining TBX4 structure and function essential for airway branching during early stages of human lung development. Substitutions of this residue may act in a dominant negative manner, leading to AcDys and CAD.

Abstract 4120919: Cell physiology and multi-omics analysis revealed disordered embryogenesis as early as mesoderm specification in models of Holt-Oram Syndrome

Introduction: Congenital heart diseases (CHDs) are the leading cause of childhood morbidity and mortality. The dysregulation of several cardiac transcription factors (TFs) leads to CHD. The coordination of several cardiac TFs is required and essential for cardiogenesis. However, the mechanisms to acquire its cardiac cell identity remain unclear. Hypothesis: Mutant Tbx5 dysregulates embryogenesis during mesoderm specification, prior to its expression in the tissues in Holt-Oram syndrome. Methods: Using cellular physiology and multiomics analysis in both human ES cells and a zebrafish model of TBX5 germline mutation. we evaluated embryonic structure and function, prior to the onset of gastrulation in the context of both heterozygous and homozygous TBX5 mutation. Results: Zebrafish time course transcriptome profiles over gastrulation revealed that loss of Tbx5 impacted transcriptional profiles at the blastula stage. Single cell RNA sequencing (scRNA-seq) on 16243 zebrafish blastula stage cells showed loss of Tbx5 impacted transcription noise at the blastula stage prior to the expression of zygotic Tbx5 with associated effects on chromatin accessibility using omni-assay for transposase-accessible chromatin (ATAC) and evidence of aberrant Wnt signaling even at the blastula stage. Embryo-wide cell structure and function were abnormal in both Tbx5 mutant zebrafish heterozygotes or homozygotes. To validate Undifferentiated human ES cell H3K4me3 Cleavage Under Targets&Release Using Nuclease (CUT&RUN) also showed abnormal Wnt signaling in TBX5 homozygous mutation and TBX5 CUT&RUN showed aberrant mesoderm pathway. Calcium imaging analysis demonstrated the lowest excitation frequency in TBX5 homozygous mutation prior to mesoderm specification. TBX5 homozygous human ES derived mesoderm cells exhibited low expression levels of mesoderm marker genes compared to TBX5 wild type mesoderm. Conclusion: Integrating single cell physiology and multi-omics technologies, we idneifified fundamental dysregulation of embryogenesis in mutant cardiac-restricted gene disorder prior to mesoderm specification or the zygotic expression of the mutant gene. These findings suggest that Tbx5 started to determine cell fate prior to mesoderm specification by altering chromatin accessibilities and histone modification. Tbx5 affected mesoderm differentiation by influencing Wnt signaling and cell physiology.

Efficient generation of liver sinusoidal endothelial-like cells secreting coagulation factor VIII from human induced pluripotent stem cells

Liver sinusoidal endothelial cells (LSECs) and LSEC progenitor cells (LPCs) derived from human pluripotent stem cells (PSCs) are expected as valuable cell sources for the development of cell therapy for hemophilia A, a congenital deficiency of coagulation factor VIII (FVIII), as LSECs are responsible for FVIII production. However, there is room for improvement in the efficiency of LSECs and LPCs differentiation from PSCs. In this study, we sought to optimize the method of mesoderm differentiation induction, the initial step of LSEC differentiation from PSCs, to efficiently induce LSEC-like cells capable of secreting FVIII from human induced pluripotent stem cells (iPSCs). Following optimization of the concentration and stimulation period of CHIR99021 (glycogen synthase kinase 3β inhibitor), bone morphogenetic protein 4, fibroblast growth factor 2, and Activin A in the mesoderm induction step, approximately 65% and 54% of cells differentiated into LPCs and LSEC-like cells, respectively. Furthermore, we observed substantial FVIII protein secretion from LSEC-like cells in vitro. In conclusion, we established an efficient method for obtaining LPCs and functional LSEC-like cells from iPSCs in vitro.

Fetal Liver-like Organoids Recapitulate Blood-Liver Niche Development and Multipotent Hematopoiesis from Human Pluripotent Stem Cells.

The fetal liver is a hematopoietic organ, hosting a diverse and evolving progenitor population. While human liver organoids derived from pluripotent stem cells (PSCs) mimic aspects of embryonic and fetal development, they typically lack the complex hematopoietic niche and the interaction between hepatic and hematopoietic development. We describe the generation of human Fetal Liver-like Organoids (FLOs), that model human hepato-hematopoietic interactions previously characterized in mouse models. Developing FLOs first integrate a yolk sac-like hemogenic endothelium into hepatic endoderm and mesoderm specification. As the hepatic and hematopoietic lineages differentiate, the FLO culture model establishes an autonomous niche capable of driving subsequent progenitor differentiation without exogenous factors. Consistent with yolk sac-derived waves, hematopoietic progenitor cells (HPCs) within FLOs exhibit multipotency with a preference for myeloid lineage commitment, while retaining fetal B and T cell differentiation potential. We reconstruct in FLOs the embryonic monocyte-to-macrophage and granulocyte immune trajectories within the FLO microenvironment and assess their functional responses in the liver niche. In vivo, FLOs demonstrate a liver engraftment bias of hematopoietic cells, recapitulating a key phenomenon of human hematopoietic ontogeny. Our findings highlight the intrinsic capacity of liver organoids to support hematopoietic development, establishing FLOs as a platform for modeling and manipulating human blood-liver niche interactions during critical stages of development and disease.

An unexpected role of IL10 in mesoderm induction and differentiation from pluripotent stem cells: Implications in zebrafish angiogenic sprouting, vascular organoid development, and therapeutic angiogenesis

Mesoderm induction is a crucial step for vascular cell specification, vascular development and vasculogenesis. However, the cellular and molecular mechanisms underlying mesoderm induction remain elusive. In the present study, a chemically-defined differentiation protocol was used to induce mesoderm formation and generate functional vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs). Zebrafish larvae were used to detect an in vivo function of interleukin 10 (IL10) in mesoderm formation and vascular development. A three dimensional approach was used to create hiPSC-derived blood vessel organoid (BVO) and explore a potential impact of IL10 on BVO formation. A murine model hind limb ischemia was applied to investigate a therapeutic potential of hiPSC-derived cells treated with or without IL10 during differentiation. We found that IL10 was significantly and specifically up-regulated during mesoderm stage of vascular differentiation. IL10 addition in mesoderm induction media dramatically increased mesoderm induction and vascular cell generation from hiPSCs, whereas an opposite effect was observed with IL10 inhibition. Mechanistic studies revealed that IL10 promotes mesoderm formation and vascular cell differentiation by activating signal transducer and activator of transcription 3 signal pathway. Functional studies with an in vivo model system confirmed that knockdown of IL10 using morpholino antisense oligonucleotides in zebrafish larvae caused defective mesoderm formation, angiogenic sprouting and vascular development. Additionally, our data also show IL10 promotes blood vessel organoid development and enhances vasculogenesis and angiogenesis. Importantly, we demonstrate that IL10 treatment during mesoderm induction stage enhances blood flow perfusion recovery and increases vasculogenesis and therapeutic angiogenesis after hind limb ischemia. Our data, therefore, demonstrate a regulatory role for IL10 in mesoderm formation from hiPSCs and during zebrafish vascular development, providing novel insights into mesoderm induction and vascular cell specifications.

Abstract We029: Common and Divergent Cellular Etiologies Underlying Hypoplastic Left Heart Syndrome and Hypoplastic Right Heart Syndrome

Single ventricle heart defects are the most severe forms of congenital heart defects and are classified based on the affected ventricles: hypoplastic left heart syndrome (HLHS) and hypoplastic right heart syndrome (HRHS). Cellular etiologies underlying differential left and right ventricular hypoplasia are unknown and remain intractable without an experimental model to probe common and divergent cellular etiologies between HLHS and HRHS. In this study, we leveraged patient-specific induced pluripotent stem cells (iPSCs) and single-cell transcriptomics to interrogate distinct etiologies of HLHS and HRHS. Both HLHS and HRHS iPSC-derived cardiomyocytes (iPSC-CMs) exhibit a reduced proliferation capacity compared to sex-matched family controls under both static and cyclic stretch conditions. Biological pathways related to cell cycle progression, DNA replication, and cell proliferation are downregulated in both HLHS and HRHS iPSC-CMs, suggesting an intrinsic cardiac proliferation deficiency. Single-cell transcriptomics indicate that differentiation of cardiac mesoderm towards second heart field (SHF) progenitors are compromised in both HLHS and HRHS. However, differentiation of epicardial progenitors is enhanced at the expense of SHF progenitors in HLHS, whereas first heart field (FHF) progenitors are prevalent in HRHS. Trajectory inference analysis uncovers distinct cell lineage determination pathways in FHF and SHF progenitors between HLHS and HRHS. Moreover, HLHS iPSC-CMs exhibit reduced mitochondrial activities whereas HRHS iPSC-CMs show enhanced mitochondrial respiration and ATP production compared to controls. In sum, these common and divergent cellular etiologies of HLHS and HRHS may underlie ventricular hypoplasia in the left side and right side of the heart.

PEG 300 Promotes Mesodermal Differentiation in iPSC-Derived Embryoid Body Formation In Vitro.

Embryoid bodies (EB) are sensitive to changes in the culture conditions. Recent studies show that the addition of PEG 300 to culture medium affects cell growth and differentiation; however, its effect on the embryoid body is unclear. This study aims to understand the role of PEG 300 in the process of EB formation and germ layer differentiation. EBs formed more efficiently and differentiated toward the mesoderm when cultured in a medium supplemented with appropriate concentrations of PEG 300. The expression of T/Bry, a marker of mesodermal differentiation, increases in EBs in the PEG group, and the expression of TUBB3 generally decreases, showing a quantitative relationship with PEG. Furthermore, further differentiation of PEG-pretreated EB into vascular smooth muscle cells (VSMCs) by directional induction shows that PEG 300-pretreated induced VSMCs have higher expression of phenotypic markers and greater secretory and contractile functions. This study highlights the role of PEG 300 in the culture medium during EB differentiation, which can significantly enhance mesodermal gene expression and the efficiency of subsequent differentiation into smooth muscle cells and other target cells.

Unraveling radiation-induced skeletal muscle damage: Insights from a 3D human skeletal muscle organoid model

BackgroundThree-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to replicate physiological tissue organization and functionality. This study investigates the impact of radiation on skeletal muscle response using an innovative in vitro human 3D skeletal muscle organoids (hSMOs) model derived from hPSCs. MethodsThe hSMOs model was established through a differentiation protocol faithfully recapitulating embryonic myogenesis and maturation via paraxial mesodermal differentiation of hPSCs. Key skeletal muscle characteristics were confirmed using immunofluorescent staining and RT-qPCR. Subsequently, the hSMOs were exposed to a clinically relevant dose of 2 Gy of radiation, and their response was analyzed using immunofluorescent staining and RNA-seq. ResultsThe hSMO model faithfully recapitulated embryonic myogenesis and maturation, maintaining key skeletal muscle characteristics. Following exposure to 2 Gy of radiation, histopathological analysis revealed deficits in hSMOs expansion, differentiation, and repair response across various cell types at early (30 min) and intermediate (18 h) time points post-radiation. Immunofluorescent staining targeting γH2AX and 53BP1 demonstrated elevated levels of foci per cell, particularly in PAX7+ cells, during early and intermediate time points, with a distinct kinetic pattern showing a decrease at 72 h. RNA-seq data provided comprehensive insights into the DNA damage response within the hSMOs. ConclusionsOur findings highlight deficits in expansion, differentiation, and repair response in hSMOs following radiation exposure, enhancing our understanding of radiation effects on skeletal muscle and contributing to strategies for mitigating radiation-induced damage in this context.

Engineering a computable epiblast for in silico modeling of developmental toxicity

Developmental hazard evaluation is an important part of assessing chemical risks during pregnancy. Toxicological outcomes from prenatal testing in pregnant animals result from complex chemical-biological interactions, and while New Approach Methods (NAMs) based on in vitro bioactivity profiles of human cells offer promising alternatives to animal testing, most of these assays lack cellular positional information, physical constraints, and regional organization of the intact embryo. Here, we engineered a fully computable model of the embryonic disc in the CompuCell3D.org modeling environment to simulate epithelial-mesenchymal transition (EMT) of epiblast cells and self-organization of mesodermal domains (chordamesoderm, paraxial, lateral plate, posterior/extraembryonic). Mesodermal fate is modeled by synthetic activity of the BMP4-NODAL-WNT signaling axis. Cell position in the epiblast determines timing with respect to EMT for 988 computational cells in the computer model. An autonomous homeobox (Hox) clock hidden in the epiblast is driven by WNT-FGF4-CDX signaling. Executing the model renders a quantitative cell-level computation of mesodermal fate and consequences of perturbation based on known biology. For example, synthetic perturbation of the control network rendered altered phenotypes (cybermorphs) mirroring some aspects of experimental mouse embryology, with electronic knockouts, under-activation (hypermorphs) or over-activation (hypermorphs) particularly affecting the size and specification of the posterior mesoderm. This foundational model is trained on embryology but capable of performing a wide variety of toxicological tasks conversing through anatomical simulation to integrate in vitro chemical bioactivity data with known embryology. It is amenable to quantitative simulation for probabilistic prediction of early developmental toxicity.

In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

Short preconditioning with TGFβ of equine adipose tissue-derived mesenchymal stem cells predisposes towards an anti-fibrotic secretory phenotype: A possible tool for treatment of endometrosis in mares

Endometrosis in mares is a disease resulting from chronic inflammation characterized by peri glandular fibrosis. There is no effective treatment so far, which opens the door for exploring the use of stem cells as a candidate. Transforming growth factor beta (TGFβ) is crucial for the establishment and progression of fibrosis in mare's endometrosis. We aimed to develop regenerative approaches to treat endometrosis by using mesenchymal stem cells (MSC), for which understanding the effect of TGFβ on exogenous MSC is crucial. We isolated and characterized equine adipose MSC from six donors. Cells were pooled and exposed to 10 ng/ml of TGFβ for 0, 4, and 24 h, after which cells were analyzed for proliferation, migration, mesodermal differentiation, expression of fibrosis-related mRNAs, and prostaglandin E2 secretion. At 24 h of exposition to TGFβ, there was a progressive increase in the contraction of the monolayer, leading to nodular structures, while cell viability did not change. Exposure to TGFβ impaired adipogenic and osteogenic differentiation after 4 h of treatment, which was more marked at 24 h, represented by a decrease in Oil red and Alizarin red staining, as well as a significant drop (p < 0.05) in the expression of key gene regulators of differentiation processes (PPARG for adipose and RUNX2 for osteogenic differentiation). TGFβ increased chondrogenic differentiation as shown by the upsurge in size of the resulting 3D cell pellet and intensity of Alcian Blue staining, as well as the significant up-regulation of SOX9 expression (p < 0.05) at 4 h, which reached a maximum peak at 24 h (p < 0.01), indicative of up-regulation of glycosaminoglycan synthesis. Preconditioning MSC with TGFβ led to a significant increase (p < 0.05) in the expression of myofibroblast gene markers aSMA, COL1A1, and TGFβ at 24 h exposition time. In contrast, the expression of COL3A1 did not change with respect to the control but registered a significant downregulation compared to 4 h (p < 0.05). TGFβ also affected the expression of genes involved in PGE2 synthesis and function; COX2, PTGES, and the PGE2 receptor EP4 were all significantly upregulated early at 4 h (p < 0.05). Cells exposed to TGFβ showed a significant upregulation of PGE2 secretion at 4 h compared to untreated cells (p < 0.05); conversely, at 24 h, the PGE2 values decreased significantly compared to control cells (p < 0.05). Preconditioning MSC for 4 h led to an anti-fibrotic secretory phenotype, while a longer period (24 h) led to a pro-fibrotic one. It is tempting to propose a 4-h preconditioning of exogenous MSC with TGFβ to drive them towards an anti-fibrotic phenotype for cellular and cell-free therapies in fibrotic diseases such as endometrosis of mares.

Inhibition of BMP-mediated SMAD pathway supports the pluripotency of pig embryonic stem cells in the absence of feeder cells

Here, we examined the effects of the BMP signaling pathway inhibitor LDN-193189 on the pluripotency of porcine embryonic stem cells (ESCs) in the absence of feeder cells using molecular and transcriptomic techniques. Additionally, the effects of some extracellular matrix components on porcine ESC pluripotency were evaluated to develop an optimized and sustainable feeder-free culture system for porcine ESCs. Feeder cells were found to play an important role in supporting the pluripotency of porcine ESCs by blocking trophoblast and mesodermal differentiation through the inhibition of the BMP pathway. Additionally, treatment with LDN-193189, an inhibitor of the BMP pathway, maintained the pluripotency and homogeneity of porcine ESCs for an extended period in the absence of feeder cells by stimulating the secretion of chemokines and suppressing differentiation, based on transcriptome analysis. Conclusively, these results suggest that LDN-193189 could be a suitable replacement for feeder cells in the maintenance of porcine ESC pluripotency during culture. Additionally, these findings contribute to the understanding of pluripotency gene networks and comparative embryogenesis.

Persistent Mesodermal Differentiation Capability of Bone Marrow MSCs Isolated from Aging Patients with Low-Energy Traumatic Hip Fracture and Osteoporosis: A Clinical Evidence.

A low-energy hit, such as a slight fall from a bed, results in a bone fracture, especially in the hip, which is a life-threatening risk for the older adult and a heavy burden for the social economy. Patients with low-energy traumatic bone fractures usually suffer a higher level of bony catabolism accompanied by osteoporosis. Bone marrow-derived stem cells (BMSCs) are critical in osteogenesis, leading to metabolic homeostasis in the healthy bony microenvironment. However, whether the BMSCs derived from the patients who suffered osteoporosis and low-energy traumatic hip fractures preserve a sustained mesodermal differentiation capability, especially in osteogenesis, is yet to be explored in a clinical setting. Therefore, we aimed to collect BMSCs from clinical hip fracture patients with osteoporosis, followed by osteogenic differentiation comparison with BMSCs from healthy young donors. The CD markers identification, cytokines examination, and adipogenic differentiation were also evaluated. The data reveal that BMSCs collected from elderly osteoporotic patients secreted approximately 122.8 pg/mL interleukin 6 (IL-6) and 180.6 pg/mL vascular endothelial growth factor (VEGF), but no PDGF-BB, IL-1b, TGF-b1, IGF-1, or TNF-α secretion. The CD markers and osteogenic and adipogenic differentiation capability in BMSCs from these elderly osteoporotic patients and healthy young donors are equivalent and compliant with the standards defined by the International Society of Cell Therapy (ISCT). Collectively, our data suggest that the elderly osteoporotic patients-derived BMSCs hold equivalent differentiation and proliferation capability and intact surface markers identical to BMSCs collected from healthy youth and are available for clinical cell therapy.

A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient’s ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids

BackgroundInduced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.MethodsDifferentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.ResultsWe observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.ConclusionOur data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Combinatorial interpretation of BMP and WNT controls the decision between primitive streak and extraembryonic fates

BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that control self-organized patterning. As development is highly dynamic, it is crucial to understand how time-dependent combinatorial signaling affects cellular differentiation. Here, we show that BMP signaling duration is a crucial control parameter that determines cell fates upon the exit from pluripotency through its interplay with the induced secondary signal WNT. BMP signaling directly converts cells from pluripotent to extraembryonic fates while simultaneously upregulating Wnt signaling, which promotes primitive streak and mesodermal specification. Using live-cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this circuit produces a temporal morphogen effect where, once BMP signal duration is above a threshold for differentiation, intermediate and long pulses of BMP signaling produce specification of mesoderm and extraembryonic fates, respectively. Our results provide a systems-level picture of how these signaling pathways control the landscape of early human development.

Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

The migration pattern of cells during the mesoderm and endoderm differentiation from human pluripotent stem cells.

Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis.