Mesodermal Commitment Research Articles

Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

The embryonic cell surface is rich in glycosphingolipids (GSLs), which change during differentiation. The reasons for GSL subgroup variation during early embryogenesis remain elusive. By combining genomic approaches, flow cytometry, confocal imaging, and transcriptomic data analysis, we discovered that α1,2-fucosylated GSLs control the differentiation of human pluripotent cells (hPCs) into germ layer tissues. Overexpression of α1,2-fucosylated GSLs disrupts hPC differentiation into mesodermal lineage and reduces differentiation into cardiomyocytes. Conversely, reducing α1,2-fucosylated groups promotes hPC differentiation and mesoderm commitment in response to external signals. We find that bone morphogenetic protein 4 (BMP4), a mesodermal gene inducer, suppresses α1,2-fucosylated GSL expression. Overexpression of α1,2-fucosylated GSLs impairs SMAD activation despite BMP4 presence, suggesting α-fucosyl end groups as BMP pathway regulators. Additionally, the absence of α1,2-fucosylated GSLs in early/late mesoderm and primitive streak stages in mouse embryos aligns with the hPC results. Thus, α1,2-fucosylated GSLs may regulate early cell-fate decisions and embryo development by modulating cell signaling.

A primate-specific endogenous retroviral envelope protein sequesters SFRP2 to regulate human cardiomyocyte development

Endogenous retroviruses (ERVs) occupy a significant part of the human genome, with some encoding proteins that influence the immune system or regulate cell-cell fusion in early extra-embryonic development. However, whether ERV-derived proteins regulate somatic development is unknown. Here, we report a somatic developmental function for the primate-specific ERVH48-1 (SUPYN/Suppressyn). ERVH48-1 encodes a fragment of a viral envelope that is expressed during early embryonic development. Loss of ERVH48-1 led to impaired mesoderm and cardiomyocyte commitment and diverted cells to an ectoderm-like fate. Mechanistically, ERVH48-1 is localized to sub-cellular membrane compartments through a functional N-terminal signal peptide and binds to the WNT antagonist SFRP2 to promote its polyubiquitination and degradation, thus limiting SFRP2 secretion and blocking repression of WNT/β-catenin signaling. Knockdown of SFRP2 or expression of a chimeric SFRP2 with the ERVH48-1 signal peptide rescued cardiomyocyte differentiation. This study demonstrates how ERVH48-1 modulates WNT/β-catenin signaling and cell type commitment in somatic development.

Alternative polyadenylation and dynamic 3' UTR length is associated with polysome recruitment throughout the cardiomyogenic differentiation of hESCs.

Alternative polyadenylation (APA) increases transcript diversity through the generation of isoforms with varying 3' untranslated region (3' UTR) lengths. As the 3' UTR harbors regulatory element target sites, such as miRNAs or RNA-binding proteins, changes in this region can impact post-transcriptional regulation and translation. Moreover, the APA landscape can change based on the cell type, cell state, or condition. Given that APA events can impact protein expression, investigating translational control is crucial for comprehending the overall cellular regulation process. Revisiting data from polysome profiling followed by RNA sequencing, we investigated the cardiomyogenic differentiation of pluripotent stem cells by identifying the transcripts that show dynamic 3' UTR lengthening or shortening, which are being actively recruited to ribosome complexes. Our findings indicate that dynamic 3' UTR lengthening is not exclusively associated with differential expression during cardiomyogenesis but rather with recruitment to polysomes. We confirm that the differentiated state of cardiomyocytes shows a preference for shorter 3' UTR in comparison to the pluripotent stage although preferences vary during the days of the differentiation process. The most distinct regulatory changes are seen in day 4 of differentiation, which is the mesoderm commitment time point of cardiomyogenesis. After identifying the miRNAs that would target specifically the alternative 3' UTR region of the isoforms, we constructed a gene regulatory network for the cardiomyogenesis process, in which genes related to the cell cycle were identified. Altogether, our work sheds light on the regulation and dynamic 3' UTR changes of polysome-recruited transcripts that take place during the cardiomyogenic differentiation of pluripotent stem cells.

The Role of Aplnr Signaling in the Developmental Regulation of Mesenchymal Stem Cell Differentiation from Human Pluripotent Stem Cells.

Stem cells are invaluable resources for personalized medicine. Mesenchymal stem cells (MSCs) have received great attention as therapeutic tools due to being a safe, ethical, and accessible option with immunomodulatory and controlled differentiation properties. Apelin receptor (Aplnr) signaling is reported to be involved in biological events, including gastrulation, mesoderm migration, proliferation of MSCs. However, the knowledge about the exact role and mechanism of Aplnr signaling during mesoderm and MSCs differentiation is still primitive. The current study aims to unveil the role of Aplnr signaling during mesoderm and MSC differentiation from pluripotent stem cells (PSCs) through peptide/small molecule activation, overexpression, knock down or CRISPR/Cas9 mediated knock out of the pathway components. Morphological changes, gene and protein expression analysis, including antibody array, LC/MS, mRNA/miRNA sequencing, reveal that Aplnr signaling promotes mesoderm commitment possibly via EGFR and TGF-beta signaling pathways and enhances migration of cells during mesoderm differentiation. Moreover, Aplnr signaling positively regulates MSCs differentiation from hPSCs and increases MSC characteristics and differentiation capacity by regulating pathways, such as EGFR, TGFβ, Wnt, PDGF, and FGF. Osteogenic, chondrogenic, adipogenic, and myogenic differentiations are significantly enhanced with Aplnr signaling activity. This study generates an important foundation to generate high potential MSCs from PSCs to be used in personalized cell therapy.

FRI686 Phenotypic Profiling And Molecular Mechanisms In Hyperparathyroidism-jaw Tumor Syndrome

Abstract Disclosure: R. Tora: None. J. Welch: None. J. Sun: None. S.K. Agarwal: None. D.A. Bell: None. M. Merino: None. L.S. Weinstein: None. W.F. Simonds: None. S. Jha: None. Background: Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome is a heritable form of primary hyperparathyroidism (PHPT) that is associated with tumors in the parathyroid, jaw, kidney, and uterus. We previously presented on the clinical characteristics of primary hyperparathyroidism in our cohort. Methods: We now expand on the report through review of uterine tumors, immunohistochemical staining for parafibromin, sequencing of parathyroid and kidney tumor tissue, and RNA-seq of parathyroid tumors. Results: We identified 68 patients (36 males, 32 females) from 29 kindreds with HPT-JT with median age at last follow-up of 39 [29-53] years. Of these, 7 patients were unaffected (2 males, 5 females). Among females, 12/32 (38%) developed uterine tumors with multiple adenomyomatous polyps, an unusual form of endometrial polyps seen in 10/24 tumors (42%). Given that these types of lesions are very rare in the general population, it is likely that such epithelial-mesenchymal uterine proliferations are a characteristic feature of HPT-JT syndrome.Solid kidney tumors were seen in 4/61 patients (7%). Three had surgery for the tumors - 2/3 were female and had mixed epithelial stromal tumor, while the third patient, a 6-year old male, had metastatic Wilm’s tumor as his initial disease-related manifestation. Among these, two had a germline CDC73 variant at the p.M1 residue while the third patient had the germline variant p.L95P; however, sequencing of his tumor tissue showed a “second hit” at the p.M1 residue resulting in loss of heterozygosity in the tumor. The fourth patient, a female with p.Y55S variant, is being conservatively managed with no kidney surgery yet. All three women with kidney tumors also had a history of uterine tumors. Parafibromin staining of 19 available CDC73 related parathyroid tumors (13 adenomas and 6 carcinoma) did not correlate with histology or genotype. Pathway analysis of 21 parathyroid tumors (8 HPT-JT-related adenomas, 6 HPT-JT-related carcinomas and 7 sporadic parathyroid carcinoma with wild-type CDC73) showed a significant association of HPT-JT-related parathyroid carcinoma with growth, transmembrane receptor protein tyrosine kinase signaling and mesodermal commitment pathways. Conclusion: Multiple, recurrent atypical adenomyomatous uterine polyps appear to be characteristic of HPT-JT syndrome. Our findings support germline testing at a young age in first-degree relatives of patients with HPT-JT given onset of manifestations as early as 6 years. Variants at M1 residue appear to predispose to genitourinary tumors. At present, there are no therapeutic approaches to compensate for CDC73 inactivation. Future research to identify standards of care and therapeutics targets are warranted for this rare disease. Presentation: Friday, June 16, 2023

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells

BackgroundIn vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs.MethodsIn this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation.ResultsAlthough YAP is dispensable for maintaining the self-renewal and pluripotency of these hiPSCs, it affects the early cell-fate determination and hematopoietic differentiation of hiPSCs. Depleting YAP enhances the derivation efficiency of HSPCs from hiPSCs by inducing the mesodermal lineage commitment, promoting hematopoietic differentiation, and preventing the differentiation toward endothelial lineage. On the contrary, the overexpression of YAP reduced HSPCs yield by inducing the endodermal lineage commitment, suppressing hematopoietic differentiation, and promoting the differentiation toward endothelial lineage.ConclusionsExpression of YAP is crucial for the differentiation of hiPSC-derived HSPCs toward mature erythrocytes. We believe that by manipulating YAP activity using small molecules, the efficiency of the large-scale in vitro production system for generating hematopoietic stem/progenitor cells for future therapeutic use could be improved.

Phenotypic Profiling and Molecular Mechanisms in Hyperparathyroidism-jaw Tumor Syndrome.

Hyperparathyroidism-jaw tumor (HPT-JT) syndrome is a heritable form of primary hyperparathyroidism caused by germline inactivating mutations in CDC73 encoding parafibromin and is associated with an increased risk of parathyroid cancer. There is little evidence to guide the management of patients with the disease. (1) Characterize the natural history of HPT-JT, (2) correlate genotype and histology of parathyroid tumors with parafibromin immunostaining, (3) understand molecular changes downstream to CDC73 loss. Retrospective study of patients with HPT-JT syndrome (genetically confirmed or affected first-degree relatives). Independent review of uterine tumor from 2 patients and staining for parafibromin on parathyroid tumors from 19 patients (13 adenomas, 6 carcinomas) was performed. RNA-sequencing was performed in 21 parathyroid samples (8 HPT-JT-related adenomas, 6 HPT-JT-related carcinomas, and 7 sporadic carcinomas with wild-type CDC73). We identified 68 patients from 29 kindreds with HPT-JT with median age at last follow-up of 39 [interquartile range, 29-53] years. A total of 55/68 (81%) developed primary hyperparathyroidism; 17/55 (31%) had parathyroid carcinoma. Twelve of 32 (38%) females developed uterine tumors. Of the 11 patients who had surgical resection for uterine tumors, 12/24 (50%) tumors were rare mixed epithelial mesenchymal polypoid lesions. Four of 68 patients (6%) developed solid kidney tumors; 3/4 had a CDC73 variant at p.M1 residue. Parafibromin staining of parathyroid tumors did not correlate with tumor histology or genotype. RNA-sequencing showed a significant association of HPT-JT-related parathyroid tumors with transmembrane receptor protein tyrosine kinase signaling pathway, mesodermal commitment pathway, and cell-cell adhesion. Multiple, recurrent atypical adenomyomatous uterine polyps appear to be enriched in women with HPT-JT and appear characteristic of the disease. Patients with CDC73 variants at p.M1 residue appear predisposed to kidney tumors. NCT04969926.

Platr4 is an early embryonic lncRNA that exerts its function downstream on cardiogenic mesodermal lineage commitment.

The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs), many of which are developmentally regulated and differentially expressed across tissues, suggesting their potential roles in cellular differentiation. Despite this expression pattern, little is known about how lncRNAs influence lineage commitment at the molecular level. Here, we demonstrate that perturbation of an embryonic stem cell/early embryonic lncRNA, pluripotency-associated transcript 4 (Platr4), directly influences the specification of cardiac-mesoderm-lineage differentiation. We show that Platr4 acts as a molecular scaffold or chaperone interacting with the Hippo-signaling pathway molecules Yap and Tead4 to regulate the expression of a downstream target gene, Ctgf, which is crucial to the cardiac-lineage program. Importantly, Platr4 knockout mice exhibit myocardial atrophy and valve mucinous degeneration, which are both associated with reduced cardiac output and sudden heart failure. Together, our findings provide evidence that Platr4 is required in cardiac-lineage specification and adult heart function in mice.

Cardiac Development in the Presence of Cadmium: An in Vitro Study Using Human Embryonic Stem Cells and Cardiac Organoids.

Exposure to cadmium (Cd) is associated with cardiovascular diseases. Maternal Cd exposure is a significant risk factor for congenital heart disease. However, mechanisms of Cd on developmental cardiotoxicity are not well defined. We evaluated the effects of Cd on the different stages (mesoderm, cardiac induction, cardiac function) of cardiac development using an early embryo development in vitro model and two- or three-dimensional (2- or 3D) cardiomyocyte and cardiac organoid formation models mimicking early cardiac development. Embryonic stem cells (ESCs) form 3D aggregates, called embryoid bodies, that recapitulate events involved with early embryogenesis (e.g., germ layer formation). This model was used for early germ layer formation and signaling pathway identification. The 2D cardiomyocyte differentiation from the human ESCs model was used to explore the effects of Cd exposure on cardiomyocyte formation and to model mesoderm differentiation and cardiac induction, allowing us to explore different developmental windows of Cd toxicity. The 3D cardiac organoid model was used in evaluating the effects of Cd exposure on contractility and cardiac development. Cd (; ) lowered the differentiation of embryoid bodies to mesoderm via suppression of -signaling pathways. During early mesoderm induction, the mesoderm-associated transcription factors MESP1 and EOMES showed a transient up-regulation, which decreased later in the cardiac induction stage. Cd () lowered mesoderm formation and cardiac induction through suppression of the transcription factors and mesoderm marker genes HAND1, SNAI2, HOPX, and the cardiac-specific genes NKX2-5, GATA4, troponin T, and . In addition, Cd-induced histone modifications for both gene activation (H3K4me3) and repression (H3K27me3), which play vital roles in regulating mesoderm commitment markers. The effects of Cd inhibition on cardiomyocyte differentiation were confirmed in 3D cardiac organoids. In conclusion, using a human ESC-derived 2D/3D in vitro differentiation model system and cardiac organoids, we demonstrated that low-dose Cd suppressed mesoderm formation through mesoderm gene histone modification, thus inhibiting cardiomyocyte differentiation and cardiac induction. The studies provide valuable insights into cellular events and molecular mechanisms associated with Cd-induced congenital heart disease. https://doi.org/10.1289/EHP11208.

PSXIII-B-13 Investigation of Genetic Markers That may be Associated with Responses of Dairy Cows to Ketosis Treatment

Abstract The objective of this study was to investigate genetic markers associated with different responses to ketosis treatment when compared to ketosis-free Holstein cows. Holstein cows (N=964) from four commercial farms, located in New York State/USA, were included in a randomized controlled trial for ketosis treatment for a period of 2 months (5/19/2019 to 7/20/2019). The cows were screened for BHB (β-hydroxybutyrate ≥1.2 mmol/L) in the first two weeks postpartum (0-14 DIM). Cows with negative results (BHB < 1.2 mmol/L) in the first week (0-7 DIM) were retested in the second week of lactation (8-14 DIM), if negative at both screening tests, cows were categorized as ketosis-free (control group). The cows with positive results in the screening period (0-14 DIM) were treated as detailed in Capel et al. (2020) and reassessed in the following two weeks. According to the cow`s response to the treatment, they were classified as CURED, RECURRENT, SEVERE, and CHRONIC. Out of the 964 cows, 489 were genotyped using the 50k Illumina Bead Chip array. A mixed linear model-based genome-wide association study (GWAS) was carried out in GCTA software. The GWAS was performed for each ketosis outcome compared to the control group. Several potential genomic regions were identified on chromosomes 1, 3, 4, 7, 8, 14, and 17. Overall, the highlighted genes included COL1A1, NEDD4l, DIP2A, CARD6, ITGA7, and MMP19, which have shared functions with genes previously associated with ketosis (e.g., CASP9, ACACA, and Interleukins) in pathways such as the transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds, Cell adhesion_ECM remodeling Pathway, TGF-beta signaling, Nucleotide-binding Oligomerization Domain (NOD), and Mesodermal commitment. These findings contribute to a better understanding of the mechanisms that trigger response to ketosis treatment and warrant further investigation.

Human mesenchymal amniotic fluid stem cells reveal an unexpected neuronal potential differentiating into functional spinal motor neurons.

Human amniotic fluids stem cells (hAFSCs) can be easily isolated from the amniotic fluid during routinely scheduled amniocentesis. Unlike hiPSCs or hESC, they are neither tumorigenic nor immunogenic and their use does not rise ethical or safety issues: for these reasons they may represent a good candidate for the regenerative medicine. hAFSCs are generally considered multipotent and committed towards the mesodermal lineages; however, they express many pluripotent markers and share some epigenetic features with hiPSCs. Hence, we hypothesized that hAFSCs may overcome their mesodermal commitment differentiating into to ectodermal lineages. Here we demonstrated that by the sequential exposure to specific factors, hAFSCs can give rise to spinal motor neurons (MNs), as evidenced by the gradual gene and protein upregulation of early and late MN markers (PAX6, ISL1, HB9, NF-L, vAChT). When co-cultured with myotubes, hAFSCs-derived MNs were able to create functional neuromuscular junctions that induced robust skeletal muscle contractions. These data demonstrated the hAFSCs are not restricted to mesodermal commitment and can generate functional MNs thus outlining an ethically acceptable strategy for the study and treatment of the neurodegenerative diseases.

Three-dimensional cardiac organoid formation accelerates the functional maturation of human induced pluripotent stem cell-derived cardiomyocytes

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) offer a promising source for heart regeneration, disease modeling, and drug screening. Recent developments in organoid technology have made it possible to study how hiPSC-derived CMs interact together, and this culture system mimics the tissue environment and behavior of the cardiac cells in our body. However, the similarities and differences between conventional 2-dimensional (2D) culture and 3-dimensional (3D) organoid culture systems for CM differentiation have been incompletely elucidated. To study how the individual microenvironment formed by each culture system affects the properties of CMs differentiated from hiPSCs, we conducted a comparative study between 2D monolayer and direct 3D cardiac organoid (hiCO) differentiation from hiPSCs throughout the sequential differentiation stages. Although identical differentiation cues were applied to hiPSCs, the 3D differentiation system strongly exhibited higher mesoderm commitment and cardiac induction than 2D monolayer differentiation. In the late stage of differentiation, the 3D hiCOs showed a higher frequency of a mature myofibrillar isoform switching in sarcomere structure of differentiated CMs than was observed in monolayer culture, although over 94% of cardiac troponin T-positive cells resulted at the end point of differentiation in both systems. Furthermore, the accelerated structural maturation in 3D hiCOs resulted in increased expression of cardiac-specific ion channel genes and Ca2+ transient properties, with a high signal amplitude and rapid contractility. The present study provides details surrounding the 2D and 3D culture methods for CM differentiation from iPSCs, and focuses on 3D cell culture as an improved strategy for approaching and applying cardiac maturation.

The Prominent Role of miR-942 in Carcinogenesis of Tumors.

As a family of short noncoding RNAs, MicroRNAs have been identified as possible biomarkers for cancer discovery and assist in therapy control due to their epigenetic involvement in gene expression and other cellular biological processes. In the present review, the evidence for reaching the clinical effect and the molecular mechanism of miR-942 in various kinds of cancer is amassed. Dysregulation of miR-942 amounts in different kinds of malignancies, as bladder cancer, esophageal squamous cell carcinoma, breast cancer, cervical cancer, gastric cancer, colorectal cancer, Kaposi's sarcoma, melanoma, Hepatocellular carcinoma, nonsmall-cell lung cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic ductal adenocarcinoma, renal cell carcinoma, and prostate cancer has stated a considerable increase or decrease in its level indicating its function as oncogene or tumor suppressor. MiR-942 is included in cell proliferation, migration, and invasion through cell cycle pathways, including pathways of transforming growth factor-beta signaling pathways, Wnt pathway, JAK/STAT pathway, PI3K/AKT pathway, apoptosis pathway, hippo signaling pathway, lectin pathway, interferon-gamma signaling, signaling by G-protein coupled receptor, developmental genes, nuclear factor-kappa B pathway, Mesodermal commitment pathway, and T-cell receptor signaling in cancer. An important biomarker, MiR-942 is a potential candidate for prediction in several cancers. The present investigation introduced miR-942 as a prognostic marker for early discovery of tumor progression, metastasis, and development.

Characterization of IPSCs-Derived Cardiomyocytes

Purpose Functional cardiomyocytes are instrumental to in vitro studies of myocardial diseases, understanding toxicities, and studying new therapies. Because functional cardiomyocytes in tissue culture are scarce and difficult to obtain we sought to differentiate and characterize cardiomyocytes derived from induced pluripotent stem cells (iPSCs) in our lab. Methods Induced pluripotent stem cells (iPSCs) from dermal fibroblasts obtained from a healthy donor were utilized to derive cardiomyocytes. Cells were cultured until 70-85% confluent and differentiated to cardiomyocytes using differentiation kit that contain a high dose of GSK3B inhibitor to induce mesodermal lineage commitment for one day that will result in up-regulation of endogenous BMP4 and activin/NODAL followed by WNT signaling modulation for two days for Cardiac mesodermal induction. Immunocytochemistry for NKX2-5 and TNNT2 were used to characterize differentiated cardiomyocytes. NanoString micro RNA was used to profile commercial and lab differentiated cardiomyocytes. Results Cells start beating on day eight and stayed beating for 30 days in maintenance media. Furthermore, cells were positive for two key markers of the human cardiac lineage: NKX2-5 for early cardiac mesoderm and TNNT2/cTNT for cardiomyocytes. The percentage of differentiation to cardiomyocytes were about 70% of the total iPSCs. Both commercial and lab dedifferentiated cardiomyocytes have similar contractility function. We compared micro RNA profile of our differentiated cardiomyocytes to commercial iPSCs-derived cardiomyocytes and they cluster very close to each other. However, both were clustered differently from miRNA profile of normal adult tissue cardiomyocytes. Conclusion Unlimited iPSCs-derived cardiomyocytes can be obtained in a robust and cost-effective method from fibroblasts. The cells have similar miRNA profile, molecular markers and contractile function in comparison to commercially available cardiomyocytes but have unique patterns of gene expression in comparison to non-failing adult tissue.

Terminal α1-2 Fucosyl Glycoconjugates Synthesized by FUT1: A Novel Key Regulator Function in Cell-Fate Decision During Early Development

Summary: The composition of cell surface glycoconjugates is set by controlled glycosyltransferase genes, whose function, in the context of cell fate decisions, is poorly understood. Using a human pluripotent cell (hPC)-based differentiation model, we have uncovered a critical function in the reduction of α1-2 fucosyltransferase (FUT1)—a glycosyltransferase catalyzing the synthesis of fucosylated glycoconjugates in α1-2 linkage—in promoting the acquisition of germ layer cell identity. We show that a FUT1 loss of function leads to the induction of lateral mesoderm gene expression, while a FUT1 gain of function prevents lateral mesoderm commitment and impairs endoderm and ectoderm fate specification, such that FUT1-overexpressing hESCs do not differentiate into cardiomyocyte lineages. Analysis of single-cell RNA-seq in mouse embryos (E5.5–E7.5) and embryo staining depict a spatiotemporal pattern of FUT1 activity, first in posterior and then in anterior-distal epiblast cells and later in the restricted region of the ectoderm and endoderm. Notably, fucosyltransferase-2 (FUT2), which acts similar to FUT1 and has been deemed indistinguishable from it, does not add α1-2 fucosyl residues to glycoconjugates instead of FUT1. According to molecular fate maps of known germ-layer markers, FUT1 is expressed in epiblast cells that acquired different layer potency than FUT2. These findings highlight an exclusive function of FUT1 and its synthesized α1-2 fucosylated glycoconjugates in deriving lineage specification and prompt a revision to the functionality of FUT1, in comparison to FUT2, during development.

Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells.

Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.

Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors.

Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two‐stage genome‐wide CRISPR‐knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA‐Seq profiling of the ZIC2‐mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA‐seq analysis showed the ZIC2 mutants affected the apelin receptor‐related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome‐wide unbiased CRISPR‐knockout screen to identify the key steps in human mesoderm precursor cell‐ and heart progenitor cell‐fate determination during in vitro hPSC cardiogenesis.

In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells.

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation

The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.

Mitochondrial Fusion by M1 Promotes Embryoid Body Cardiac Differentiation of Human Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) can be differentiated in vitro into bona fide cardiomyocytes for disease modelling and personalized medicine. Mitochondrial morphology and metabolism change dramatically as iPSCs differentiate into mesodermal cardiac lineages. Inhibiting mitochondrial fission has been shown to promote cardiac differentiation of iPSCs. However, the effect of hydrazone M1, a small molecule that promotes mitochondrial fusion, on cardiac mesodermal commitment of human iPSCs is unknown. Here, we demonstrate that treatment with M1 promoted mitochondrial fusion in human iPSCs. Treatment of iPSCs with M1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. The pro-fusion and pro-cardiogenic effects of M1 were not associated with changes in expression of the α and β subunits of adenosine triphosphate (ATP) synthase. Our findings demonstrate for the first time that hydrazone M1 is capable of promoting cardiac differentiation of human iPSCs, highlighting the important role of mitochondrial dynamics in cardiac mesoderm lineage specification and cardiac development. M1 and other mitochondrial fusion promoters emerge as promising molecular targets to generate lineages of the heart from human iPSCs for patient-specific regenerative medicine.