Differentiation Of Human Pluripotent Cells 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.

Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

The Role of Components of the Extracellular Matrix in the Differentiation of Human Pluripotent Cells into Ectodermal Derivatives

The Role of Components of the Extracellular Matrix in the Differentiation of Human Pluripotent Cells into Ectodermal Derivatives

SARS-CoV-2 infects an in vitro model of the human developing pancreas through endocytosis

SummaryRecent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic β-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells.

Understanding the molecular and cellular mechanisms of human reproductive development has been limited by the scarcity of human samples and ethical constraints. Recently, in vitro differentiation of human pluripotent stem cells into germ cells and single-cell analyses have opened new avenues to directly study human germ cells and identify unique mechanisms in human reproductive development. The goal of this review is to collate novel findings and insightful discoveries with these new methodologies, aiming at introducing researchers and clinicians to the use of these tools to study human reproductive biology and develop treatments for infertility. PubMed was used to search articles and reviews with the following main keywords: in vitro differentiation, human stem cells, single-cell analysis, spermatogenesis, oogenesis, germ cells and other key terms related to these subjects. The search period included all publications from 2000 until now. Single-cell analyses of human gonads have identified many important gene markers at different developmental stages and in subpopulations of cells. To validate the functional roles of these gene markers, researchers have used the in vitro differentiation of human pluripotent cells into germ cells and confirmed that some genetic requirements are unique in human germ cells and are not conserved in mouse models. Moreover, transcriptional regulatory networks and the interaction of germ and somatic cells in gonads were elucidated in these studies. Single-cell analyses allow researchers to identify gene markers and potential regulatory networks using limited clinical samples. On the other hand, in vitro differentiation methods provide clinical researchers with tools to examine these newly identify gene markers and study the causative effects of mutations previously associated with infertility. Combining these two methodologies, researchers can identify gene markers and networks which are essential and unique in human reproductive development, thereby producing more accurate diagnostic tools for assessing reproductive disorders and developing treatments for infertility.

CellSIUS provides sensitive and specific detection of rare cell populations from complex single-cell RNA-seq data

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.

Synergy with TGFβ ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation

WNT/β-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of β-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that β-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFβ superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the β-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/β-catenin dynamics.

Inhibition of MicroRNA-221 and 222 Enhances Hematopoietic Differentiation from Human Pluripotent Stem Cells via c-KIT Upregulation.

The stem cell factor (SCF)/c-KIT axis plays an important role in the hematopoietic differentiation of human pluripotent stem cells (hPSCs), but its regulatory mechanisms involving microRNAs (miRs) are not fully elucidated. Here, we demonstrated that supplementation with SCF increases the hematopoietic differentiation of hPSCs via the interaction with its receptor tyrosine kinase c-KIT, which is modulated by miR-221 and miR-222. c-KIT is comparably expressed in undifferentiated human embryonic and induced pluripotent stem cells. The inhibition of SCF signaling via treatment with a c-KIT antagonist (imatinib) during hPSC-derived hematopoiesis resulted in reductions in the yield and multi-lineage potential of hematopoietic progenitors. We found that the transcript levels of miR-221 and miR-222 targeting c-KIT were significantly lower in the pluripotent state than they were in terminally differentiated somatic cells. Furthermore, suppression of miR-221 and miR-222 in undifferentiated hPSC cultures induced more hematopoiesis by increasing c-KIT expression. Collectively, our data implied that the modulation of c-KIT by miRs may provide further potential strategies to expedite the generation of functional blood cells for therapeutic approaches and the study of the cellular machinery related to hematologic malignant diseases such as leukemia.

Specification of trophoblast from embryonic stem cells exposed to BMP4.

Trophoblast (TB) comprises the outer cell layers of the mammalian placenta that make direct contact with the maternal uterus and, in species with a highly invasive placenta, maternal blood. It has its origin as trophectoderm, a single epithelial layer of extra-embryonic ectoderm that surrounds the embryo proper at the blastocyst stage of development. Here, we briefly compare the features of TB specification and determination in the mouse and the human. We then review research on a model system that has been increasingly employed to study TB emergence, namely the BMP4 (bone morphogenetic protein-4)-directed differentiation of human embryonic stem cells (ESCd), and discuss why outcomes using it have proved so uneven. We also examine the controversial aspects of this model, particularly the issue of whether or not the ESCd represents TB at all. Our focus here has been to explore similarities and potential differences between the phenotypes of ESCd, trophectoderm, placental villous TB, and human TB stem cells. We then explore the role of BMP4 in the differentiation of human pluripotent cells to TB and suggest that it converts the ESC into a totipotent state that is primed for TB differentiation when self-renewal is blocked. Finally we speculate that the TB formed from ESC is homologous to the trophectoderm-derived, invasive TB that envelopes the implanting conceptus during the second week of pregnancy.

Exogenous TPRX1 homeoprotein modulates the gene expression of lineage-specific transcription factors in human embryonal carcinoma cells

ABSTRACTWe reported previously that EGAM1 homeoproteins (EGAM1 and EGAM1N), transcribed from the Crxos gene as splicing variants, are expressed in preimplantation mouse embryos and mouse embryonic stem (ES) cells. Exogenous expression of these proteins affects the maintenance of an undifferentiated state and the progression of differentiation in mouse ES cells. Human tetrapeptide-repeat homeobox 1 (TPRX1), a member of the eutherian totipotent cell homeobox (ETCHbox) genes, is an ortholog of Crxos. However, the roles of TPRX1 in the differentiation of human pluripotent cells are still unknown. Because the TPRX1 transcripts were undetectable in an undifferentiated state and during the progression of differentiation in wild-type human embryonal carcinoma NT2/D1 cells, it would be advantageous to clarify the relationship between the exogenous expression of TPRX1 and the induction of genes encoding lineage-specific transcription factors in pluripotent cells. The expression of GATA6 and FOXA2, crucial transcription factors for the formation of the primitive endoderm, was upregulated, whereas that of CDX2, a crucial transcription factor for the formation of the trophectoderm, was downregulated by enforced expression of TPRX1. Overall, we suggest that TPRX1 is capable of modulating the expression of lineage-specific transcription factors in pluripotent cells derived from humans.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

ABSTRACTRobust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine.

Regenerative MedicineVol. 11, No. 2 Research HighlightsOur panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicineThomas Bollenbach, Jizeng Qiao & Amber Kerstetter-FogleThomas Bollenbach Research & Development, Harvard Apparatus Regenerative Technology, 84 October Hill Road, Holliston, MA 01746, USASearch for more papers by this author, Jizeng Qiao Organogenesis, Inc., 150 Dan Rd, Canton, MA 02021, USASearch for more papers by this author & Amber Kerstetter-Fogle Case Western Reserve University, Cleveland, OH, USASearch for more papers by this authorPublished Online:29 Feb 2016https://doi.org/10.2217/rme.16.2AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleReferences1 Hiki N, Nunobe S, Kubota T et al. Function-preserving gastrectomy for early gastric cancer. Ann. Surg. Oncol. 20, 2683–2692 (2013).Crossref, Medline, Google Scholar2 Watanabe M, Shin'oka T, Tohyama S et al. Tissue-engineered vascular autograft: inferior vena cava replacement in a dog model. Tissue Eng. 7, 429–439 (2001).Crossref, Medline, CAS, Google Scholar3 Nigam SK, Wu W, Bush KT. In vitro kidney development, tissue engineering and systems biology. Organogenesis 4(3), 137–143 (2008).Crossref, Medline, Google Scholar4 Grobstein C. Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell Res. 10, 424–440 (1956).Crossref, Medline, CAS, Google Scholar5 Herzlinger D, Qiao J, Cohen D, Ramakrishna N, Brown AMC. Induction of kidney epithelial morphogenesis by cells expression Wnt-1. Dev. Biol. 166, 815–818 (1994).Crossref, Medline, CAS, Google Scholar6 Qiao J, Cohen D, Herzlinger D. The metanephric blastema differentiates into collecting system and nephron epithelial in vitro. Development 121, 3207–3214 (1995).Crossref, Medline, CAS, Google Scholar7 Qiao J, Sakurai H, Nigam SK. Branching morphogenesis independent of mesenchymal–epithelial contact in the developing kidney. Proc. Natl Acad. Sci. USA 96, 7330–7335 (1999).Crossref, Medline, CAS, Google Scholar8 Rosines E, Sampogna RV, Johkura K et al. Staged in vitro reconstitution and implantation of engineered rat kidney tissue. Proc. Natl Acad. Sci. USA 104, 20938–20943 (2007).Crossref, Medline, CAS, Google Scholar9 Song B, Smink AM, Hones CV et al. The directed differentiation of human iPS cells into kidney podocytes. PLoS ONE 7(9), e4645 (2012).Crossref, Google Scholar10 Xia Y, Nivet E, Sanchi-Martinez I et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat. Cell Biol. 15(12), 1507–1515 (2013).Crossref, Medline, CAS, Google Scholar11 Narayanan K, Schumacher KM, Tasnim F et al. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells. Kidney Int. 83, 593–603 (2013).Crossref, Medline, CAS, Google Scholar12 Takasato M, Er PX, Becroft M et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat. Cell Biol. 16, 118–126 (2014).Crossref, Medline, CAS, Google Scholar13 Araoka T, Mae S, Kurose Y et al. Efficient and rapid induction of human iPSCs/ESCs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods. PLoS ONE 9(1), e84881 (2014).Crossref, Medline, Google Scholar14 Xinaris C, Benedetti V, Rizzo P et al. In vivo maturation of functional renal organoids formed from embryonic cell suspensions. J. Am. Soc. Nephrol. 23, 1857–1868 (2012).Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Vol. 11, No. 2 Follow us on social media for the latest updates Metrics Downloaded 190 times History Published online 29 February 2016 Published in print March 2016 Information© Future Medicine LtdFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

Cortical Differentiation of Human Pluripotent Cells for In Vitro Modeling of Alzheimer's Disease.

Stem cell models of Alzheimer's disease provide an opportunity to study the mechanisms underlying disease pathology at a resolution that is not possible in animal models. Furthermore, the ability to reprogram patient somatic cells to a pluripotent state ensures that the disease can be investigated in the correct genetic context. Here, we describe the directed differentiation of human pluripotent cells to cortical progenitors by recapitulating key developmental signaling events in vitro. Over a timeframe that mirrors human development, these progenitors give rise to functional lower and upper layer neurons. We also describe biochemical and imaging based methods to analyse key APP and Tau phenotypes in neurons generated from pluripotent stem cells from individuals with either monogenic familial Alzheimer's disease or Down's syndrome.

Generating a self-organizing kidney from pluripotent cells.

Recent studies on the directed differentiation of human pluripotent stem cells report tissue self-organization in vitro such that multiple component cell types arise in concert and arrange with respect to each, thereby recapitulating the morphogenetic events typical for that organ. Such self-organization has generated pituitary, optic cup, liver, brain, intestine, stomach and now kidney. Here, we will describe the cell types present within the self-organizing kidney, how these signal to each other to form a kidney organoid and the potential applications of kidney organoids. Protocols for the directed differentiation of human pluripotent cells focus on recapitulating the developmental steps required during embryogenesis. In the case of the kidney, this has involved mesodermal differentiation through posterior primitive streak and intermediate mesoderm. Recent studies have observed the simultaneous formation of both ureteric epithelium and nephron progenitors in vitro. These component cell types signal to each other to initiate nephron formation as would occur during development. The generation of kidney organoids is a major advance in nephrology. Such organoids may be useful for disease modelling and drug screening. Ultimately, our capacity to generate organoids may extend to the development of tissues for transplantation.

Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

The generation of kidney organoids by differentiation of human pluripotent cells to ureteric bud progenitor-like cells.

This protocol presents recently developed methodologies for the differentiation of human pluripotent stem cells (hPSCs) into ureteric bud (UB) progenitor-like cells. Differentiation of human PSCs to UB progenitor-like cells allows for the generation of chimeric kidney cultures in which the human cells can self-assemble into chimeric 3D structures in combination with embryonic mouse kidney cells over a period of 18 d. UB progenitor-like cells are generated by a two-step process that combines in vitro commitment of human PSCs, whether embryonic stem cells (ESCs) or induced PSCs (iPSCs), under chemically defined culture conditions, with ex vivo cultures for the induction of 3D organogenesis. The models described here provide new opportunities for investigating human kidney development, modeling disease, evaluating regenerative medicine strategies, as well as for toxicology studies.

Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

Macro Histone Variants Are Critical for the Differentiation of Human Pluripotent Cells

We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes.