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The capacity for regeneration is broad in the animal kingdom. Planarians are flatworms that can regenerate any missing body part and their regenerative powers have combined with ease of experimentation to make them a classic regeneration model for more than a century. Pluripotent stem cells called neoblasts generate missing planarian tissues. Fate specification happens in the neoblasts, and this can occur in response to regeneration instructions in the form of positional information. Fate specification can lead to differentiating cells in single steps rather than requiring a long lineage hierarchy. Planarians display constitutive expression of positional information from muscle cells, which is required for patterned maintenance of tissues in tissue turnover. Amputation leads to the rapid resetting of positional information in a process triggered by wound signaling and the resetting of positional information is required for regeneration. These findings suggest a model for planarian regeneration in which adult positional information resets after injury to regulate stem cells to bring about the replacement of missing parts.

The 9th biennial conference titled “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology.

With the positive outcomes of various cell therapies currently under preclinical and clinical studies, there is a significant interest in novel stem cell sources with unique therapeutic properties. Studies over the past two decades or so demonstrated the feasibility to isolate multipotent/pluripotent stem cells from hair follicles. The easy accessibility, high proliferation, and differentiation ability as well as lack of ethical concerns associated with this stem cell source make hair follicle stem cells (HFSCs) attractive candidate for cell therapy and tissue engineering. This review discusses the various stem cell types identified in rodent and human hair follicles and ongoing studies on the potential use of HFSCs for skin, bone, cardiovascular, and nerve tissue engineering. Impact statement Hair follicle stem cells are an autologous stem cell source, and recent preclinical and clinical studies demonstrated its unique properties to support and accelerate tissue regeneration, making it an attractive candidate for cell therapy and tissue engineering.

Kidney organoid technology has led to a renaissance in kidney developmental biology. The complex underpinnings of mammalian kidney development have provided a framework for the generation of kidney cells and tissues from human pluripotent stem cells. Termed kidney organoids, these 3-dimensional structures contain kidney-specific cell types distributed similarly to in vivo architecture. The adult human kidney forms from the reciprocal induction of two disparate tissues, the metanephric mesenchyme (MM) and ureteric bud (UB), to form nephrons and collecting ducts, respectively. Although nephrons and collecting ducts are derived from the intermediate mesoderm (IM), their development deviates in time and space to impart distinctive inductive signaling for which separate differentiation protocols are required. Here we summarize the directed differentiation protocols which generate nephron kidney organoids and collecting duct kidney organoids, making note of similarities as much as differences. We discuss limitations of these present approaches and discuss future directions to improve kidney organoid technology, including a greater understanding of anterior IM and its derivatives to enable an improved differentiation protocol to collecting duct organoids for which historic and future developmental biology studies will be instrumental.

Pluripotent stem cells (PSCs) have unlimited capacity for self-renewal and differentiation so that they can potentially produce any cell or tissue of animal’s body. The PSCs derived from livestock represents a more appropriate model than a rodent for investigating human diseases due to their higher anatomical and physiological resemblance with human. Apart from that, livestock PSCs hold immense promises for innovative therapies, transgenic animal production and their biomedical interest. The realization of the full potential of PSCs, however, depends on the elucidation of the molecular mechanisms which play a critical role in the maintenance of pluripotency and reprogramming procedure remains poorly understood in livestock which in turn impedes the generation of true PSCs and their usage for clinical research. An in-depth understanding of pluripotency is extremely essential for improving health and welfare of livestock animals. Therefore, the present review focuses on the milestone achievements of PSCs in livestock animals and their potential application in health and production of livestock.

The endoderm, one of the three primary germ layers, gives rise to lung, liver, stomach, intestine, colon, pancreas, bladder, and thyroid. These endoderm-originated organs are subject to many life-threatening diseases. However, primary cells/tissues from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells (hPSCs), therefore, hold great promise for generating endodermal cells and their derivatives for the development of new therapeutics against these human diseases. Although a wealth of research has provided crucial information on the mechanisms underlying endoderm differentiation from hPSCs, increasing evidence has shown that metabolism, in connection with epigenetics, actively regulates endoderm differentiation in addition to the conventional endoderm inducing signals. Here we review recent advances in metabolic and epigenetic regulation of endoderm differentiation.

Human pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (31.7% of heterozygous clones, 9.1% of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs. Graphical abstract

In the past several decades, the establishment of in vitro models of pluripotency has ushered in a golden era for developmental and stem cell biology. Research in this arena has led to profound insights into the regulatory features that shape early embryonic development. Nevertheless, an integrative theory of the epigenetic principles that govern the pluripotent nucleus remains elusive. Here, we summarize the epigenetic characteristics that define the pluripotent state. We cover what is currently known about the epigenome of pluripotent stem cells and reflect on the use of embryonic stem cells as an experimental system. In addition, we highlight insights from super-resolution microscopy, which have advanced our understanding of the form and function of chromatin, particularly its role in establishing the characteristically "open chromatin" of pluripotent nuclei. Further, we discuss the rapid improvements in 3C-based methods, which have given us a means to investigate the 3D spatial organization of the pluripotent genome. This has aided the adaptation of prior notions of a "pluripotent molecular circuitry" into a more holistic model, where hotspots of co-interacting domains correspond with the accumulation of pluripotency-associated factors. Finally, we relate these earlier hypotheses to an emerging model of phase separation, which posits that a biophysical mechanism may presuppose the formation of a pluripotent-state-defining transcriptional program.

Summary Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a mystery; knockout of Dppa2 and Dppa4 did not affect pluripotency, but caused lung and skeletal defects late in development, long after Dppa2 and Dppa4 expression had been turned off. A number of recent papers have further clarified and defined the roles of these important factors, identifying roles in priming the chromatin and maintaining developmental competency through regulating both H3K4me3 and H3K27me3 at bivalent chromatin domains, and acting to remodel chromatin and facilitate reprogramming of somatic cells to induced pluripotency. These findings highlight an important regulatory role for DPPA2 and DPPA4 at the transitional boundary between pluripotency and differentiation and may have relevance to the functions of DPPA2 and 4 in the context of cancer cells as well., In this review, R. Klein and P. Knoepfler summarize the roles of DPPA family members in both pluripotency and oncogenesis, highlighting the connection between these two cellular states. They describe the functions of DPPA factors in priming the chromatin and acting at the boundary between pluripotency and differentiation.

Objectives We set out to reprogram adult somatic oral epithelial keratinocytes into pluripotent cells for regenerative dentistry. Setting and sample population Immortalized murine oral keratinocyte cell (IMOK) line raised from adult mouse mucosa were cultured in vitro in our studies. Materials and methods Adult murine oral epithelial keratinocytes were chronically treated with TGF-β1 in vitro, and the expression of Oct4, Nanog, Sox2 and Nestin, as well as specific homeobox Gata and Pax gene family members were investigated. Results We documented the induction of stem factors linked with pluripotency and/or the maintenance and regulation of stem-cell self-renewal in oral epithelial keratinocytes by TGFβ1. Moreover, we discovered that this TGF-β1-induced increase in Oct4, Nanog, Sox2 and Nestin was inhibited by SB431542, suggesting that TGF-β1 signals via the TGF-βRI receptor to induce pluripotency and stemness. Conclusions Adult oral epithelial keratinocytes treated chronically with TGF-β1 acquired phenotypic characteristics consistent with pluripotent stem cells, highlighting the facileness of reprogramming adult oral keratinocytes into an unlimited supply of pluripotent stem cells.

Organoids are three-dimensional structures that self-organize from human pluripotent stem cells or primary tissue, potentially serving as a traceable and manipulatable platform to facilitate our understanding of organogenesis. Despite the ongoing advancement in generating organoids of diverse systems, biological applications of in vitro generated organoids remain as a major challenge in part due to a substantial lack of intricate complexity. The studies of development and regeneration enumerate the essential roles of highly diversified nonepithelial populations such as mesenchyme and endothelium in directing fate specification, morphogenesis, and maturation. Furthermore, organoids with physiological and homeostatic functions require direct and indirect inter-organ crosstalk recapitulating what is seen in organogenesis. We herein review the evolving organoid technology at the cell, tissue, organ, and system level with a main emphasis on endoderm derivatives.

Summary One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro. Importantly, these cells remained highly regenerative in vivo. Upon transplantation, the expanded cells formed new DYSTROPHIN+ fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation., Graphical abstract, Highlights • Skeletal myogenic progenitors from PSC-derived teratomas are expandable in vitro • Expanded cells are PAX7+ and are capable of differentiating into MHC+ myotubes • Expanded cells engraft and regenerate fibers with adult myosins and innervation • Expanded cells repopulate the muscle stem cell niche after transplantation, In this article, Chan and colleagues show that skeletal myogenic progenitors obtained from PSC-derived teratomas are expandable in cultures and remain highly regenerative. Upon transplantation, the expanded cells robustly engraft to form new muscle fibers and reconstitute the muscle stem cell pool. This study is an advance toward generating large quantities of engraftable skeletal myogenic progenitors for downstream applications.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

The transcription factors (TFs) OCT4, SOX2 and NANOG are key players of the gene regulatory network of pluripotent stem cells. Evidence accumulated in recent years shows that even small imbalances in the expression levels or relative concentrations of these TFs affect both, the maintenance of pluripotency and cell fate decisions. In addition, many components of the transcriptional machinery including RNA polymerases, cofactors and TFs such as those required for pluripotency, do not distribute homogeneously in the nucleus but concentrate in multiple foci influencing the delivery of these molecules to their DNA-targets. How cells control strict levels of available pluripotency TFs in this heterogeneous space and the biological role of these foci remain elusive. In recent years, a wealth of evidence led to propose that many of the nuclear compartments are formed through a liquid–liquid phase separation process. This new paradigm early penetrated the stem cells field since many key players of the pluripotency circuitry seem to phase-separate. Overall, the formation of liquid compartments may modulate the kinetics of biochemical reactions and consequently regulate many nuclear processes. Here, we review the state-of-the-art knowledge of compartmentalization in the cell nucleus and the relevance of this process for transcriptional regulation, particularly in pluripotent stem cells. We also highlight the recent advances and new ideas in the field showing how compartmentalization may affect pluripotency preservation and cell fate decisions.

Human-induced pluripotent stem cell (iPSC)-derived kidney organoids have the potential to advance studies to kidney development and disease. However, reproducible generation of kidney organoids is a challenge. A large variability in the percentage of nephron structures and the expression of kidney-specific genes was observed among organoids, showing no association with iPSC lines. To associate the quality of kidney organoid differentiation with predictive markers, a ranking system was developed based on the ratio of nephron structure determined by histological examination. Well-differentiated organoids were defined as organoids with >30% nephron structure and vice versa. Subsequently, correlations were made with expression profiles of iPSC markers, early kidney development markers, and fibrosis markers. Higher expression of sex-determining region Y-box 2 (SOX2) during differentiation was associated with poorly differentiated kidney organoid. Furthermore, early secretion of basic fibroblast growth factor (FGF2) predicted poorly differentiated kidney organoid. Of interest, whereas cadherin-1 (CDH1) expression in kidney organoids indicates distal tubules formation, onefold higher CDH1 expression in iPSC predicted poor differentiation. High expression of the stromal progenitor marker Forkhead Box D1 (FOXD1) and significantly increased TGFβ levels were found in well-differentiated kidney organoids. These early expression profiles could predict the outcome of kidney organoid formation. This study helps to improve the robustness of kidney organoid protocols.

BACKGROUND The process of liver organogenesis has served as a paradigm for organ formation. However, there remains a lack of understanding regarding early mouse and human liver bud morphogenesis and early liver volumetric growth. Elucidating dynamic changes in liver volumes is critical for understanding organ development, implementing toxicological studies, and for modeling hPSC-derived liver organoid growth. New visualization, analysis, and experimental techniques are desperately needed. RESULTS Here, we combine observational data with digital resources, new 3D imaging approaches, retrospective analysis of liver volume data, mathematical modeling, and experiments with hPSC-derived liver organoids. Mouse and human liver organogenesis, characterized by exponential growth, demonstrate distinct spatial features and growth curves over time, which we mathematically modeled using Gompertz models. Visualization of liver-epithelial and septum transversum mesenchyme (STM) interactions suggests extended interactions, which together with new spatial features may be responsible for extensive exponential growth. These STM interactions are modeled with a novel in vitro human pluripotent stem cell (hPSC)-derived hepatic organoid system that exhibits cell migration. CONCLUSIONS Our methods enhance our understanding of liver organogenesis, with new 3D visualization, analysis, mathematical modeling, and in vitro models with hPSCs. Our approach highlights mouse and human differences and provides potential hypothesis for further investigation in vitro and in vivo.

BACKGROUND Diabetes mellitus (DM) is a chronic metabolic disorder, increasing in the number of patients and poses a severe threat to human health. Significant advances have been made in DM treatment; the most important of which is differentiation and proliferation of beta cells from IPSCs. METHODS Data were collected from PUBMED at various time points up to the academic year of 2020. The related keywords are listed as follows: "Induced pluripotent stem cell", "Proliferation", "Growth factor", "Small molecule", "cardiotoxicity" and "Scaffold." RESULT The use of growth factors along with small molecules can be a good strategy for beta-cell proliferation. Also, proliferation of beta cells on nanofibers scaffolds can create a similar in vivo environment, that leads to increased function of beta-cell. Some transcription factors that cause beta cells proliferation play an important role in inflammation; so, it is essential to monitor them to prevent inflammation. CONCLUSION Finally, the simultaneous use of growth factors, micronutrients and scaffolds can be an excellent strategy to increase the proliferation and function of beta cells derived from IPSCs.

Key Points Generation, identification, isolation, and expansion of monocyte and osteoclast progenitors from human embryonic stem cells.CD11b−CD14−CD115+ cells from embryonic stem-derived cultures and peripheral blood mononuclear cells contain monocyte/osteoclast progenitors., Visual Abstract, Osteoclasts are multinuclear cells of monocytic lineage, with the ability to resorb bone. Studies in mouse have identified bone marrow clonal progenitors able to generate mature osteoclast cells (OCs) in vitro and in vivo. These osteoclast progenitors (OCPs) can also generate macrophages and dendritic cells. Interestingly, cells with equivalent potential can be detected in periphery. In humans, cells with OCP activity have been identified in bone marrow and periphery; however, their characterization has not been as extensive. We have developed reproducible methods to derive, from human pluripotent stem cells, a population containing monocyte progenitors able to generate functional OCs. Within this population, we have identified cells with monocyte and osteoclast progenitor activity based on CD11b and CD14 expression. A population double positive for CD11b and CD14 contains cells with expected osteoclastic potential. However, the double negative (DN) population, containing most of the hematopoietic progenitor activity, also presents a very high osteoclastic potential. These progenitor cells can also be differentiated to macrophage and dendritic cells. Further dissection within the DN population identified cells bearing the phenotype CD15−CD115+ as the population with highest monocytic progenitor and osteoclastic potential. When similar methodology was used to identify OCPs from human peripheral blood, we confirmed a published OCP population with the phenotype CD11b+CD14+. In addition, we identified a second population (CD14−CD11bloCD115+) with high monocytic progenitor activity that was also able to form osteoclast like cells, similar to the 2 populations identified from pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease., Current protocols to generate cholangiocytes from human pluripotent cells produce immature cells. Here the authors identify retinoic acid, BMP, cAMP and Rho kinase pathways as regulators of cholangiocyte maturation, and generate ciliated cholangiocytes expressing high levels of CFTR that form ductal structures in vivo.

Summary p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice., Graphical abstract, Highlights • H3K4me defective WDR5 supports self-renewal and GCM differentiation in p53-null mESCs • WDR5 regulates H3K4me-independent stemness and GCM gene expression in p53-null mESCs • MAX and WDR5 repress GCM-related gene chromatin accessibility upon differentiation, In this article, Rao and colleagues show that an H3K4 methylation-independent, WDR5-driven mechanism maintains self-renewal and represses germ cell/meiosis (GCM)-specific transcription in p53-null mESCs. WDR5 inactivation triggers enhanced chromatin accessibility on most gene targets bound by MAX and WDR5. Thus, WDR5, a component of polycomb repressive complex 1.6, controls cell type-specific gene repression during mESC fate determination in a non-canonical fashion.

Funder: National Institute of Health (NIH) DP2-GM123495, DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome���s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) ��� sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Pluripotent cells in the mammalian embryo undergo state transitions marked by changes in patterns of gene expression and developmental potential as they progress from pre-implantation through post-implantation stages of development. Recent studies of cultured mouse and human pluripotent stem cells (hPSCs) have identified cells representative of an intermediate stage (referred to as the formative state) between naive pluripotency (equivalent to pre-implantation epiblast) and primed pluripotency (equivalent to late post-implantation epiblast). We examine these recent findings in light of our knowledge of peri-implantation mouse and human development, and we consider the implications of this work for deriving human embryo models from pluripotent cells.

Summary Tbx3 has been identified as a regulator of liver development in the mouse, but its function in human liver development remains unknown. TBX3 mutant human pluripotent stem cell (PSC) lines were generated using CRISPR/Cas9 genome editing. TBX3 loss led to impaired liver differentiation and an upregulation of pancreatic gene expression, including PDX1, during a hepatocyte differentiation protocol. Other pancreatic genes, including NEUROG3 and NKX2.2, displayed more open chromatin in the TBX3 mutant hepatoblasts. Using a pancreatic differentiation protocol, cells lacking TBX3 generated more pancreatic progenitors and had an enhanced pancreatic gene expression signature at the expense of hepatic gene expression. These data highlight a potential role of TBX3 in regulating hepatic and pancreatic domains during foregut patterning, with implications for enhancing the generation of pancreatic progenitors from PSCs., Graphical abstract, Highlights • TBX3 null PSCs have impaired hepatocyte differentiation capacity • TBX3 null hepatocytes have aberrant expression of pancreatic genes, including PDX1 • TBX3 null PSCs have enhanced differentiation capacity into pancreatic progenitors • Loss of TBX3 leads to increased chromatin accessibility of many pancreatic genes, Mukherjee et al. describe a potential role of TBX3 in patterning hepatic versus pancreatic domains during foregut development. Differentiation of TBX3 null PSCs using a hepatocyte differentiation protocol display impaired hepatocyte gene expression and upregulated expression and chromatin accessibility of pancreatic genes such as PDX1. Using a pancreatic differentiation protocol, TBX3 null PSCs have increased efficiency in pancreatic progenitor generation.

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease., Heller et al analyze transcriptomic and epigenetic datasets from human PSCs differentiating into pancreatic progenitors to elucidate the regulatory mechanisms behind pancreatic development. The authors report that the transcription factor ONECUT1 is expressed specifically in pancreatic development and associates with other key TFs (such as FOXA2, GATA6, PDX1) during endocrine specification.

Human pluripotent stem cells (PSCs), which have the capacity to self-renew and differentiate into multiple cell types, offer tremendous therapeutic potential and invaluable flexibility as research tools. Recently, remarkable progress has been made in directing myogenic differentiation of human PSCs. The differentiation strategies, which were inspired by our knowledge of myogenesis in vivo, have provided an important platform for the study of human muscle development and modelling of muscular diseases, as well as a promising source of cells for cell therapy to treat muscular dystrophies. In this review, we summarize the current state of skeletal muscle generation from human PSCs, including transgene-based and transgene-free differentiation protocols, and 3D muscle tissue production through bioengineering approaches. We also highlight their basic and clinical applications, which facilitate the study of human muscle biology and deliver new hope for muscular disease treatment.

Summary Mouse embryonic stem cells (ESCs) show cell-to-cell heterogeneity. A small number of two-cell-like cells (2CLCs) marked by endogenous retrovirus activation emerge spontaneously. The 2CLCs are unstable and they are prone to transiting back to the pluripotent state without extrinsic stimulus. To understand how this bidirectional transition takes place, we performed single-cell RNA sequencing on isolated 2CLCs that underwent 2C-like state exit and re-entry, and revealed a step-by-step transitional process between 2C-like and pluripotent states. Mechanistically, we found that cell cycle played an important role in mediating these transitions by regulating assembly of the nucleolus and peri-nucleolar heterochromatin to influence 2C gene Dux expression. Collectively, our findings provide a roadmap of the 2C-like state entry and exit in ESCs and also a causal role of the cell cycle in promoting these transitions., Highlights • The entry to and exit from the 2C-like state showed a step-by-step roadmap • Cell cycle participates in mediating dynamic transitions between ESCs and 2CLCs • G1/S phase arrest facilitates the Dux locus escape from heterochromatin • Nucleolus-heterochromatin remodeling is involved in 2C activation, In this article, Jin Zhang and colleagues show that cell-cycle variations distinguish 2CLCs, ESCs, and the intermediate states using single-cell RNA-seq. They found that cell-cycle-associated remodeling of nucleoli and peri-nucleolar heterochromatin was involved in the regulation of the 2C program. They provide novel advances in the fundamental field of 2C activation and stem cell fate decision.

Clinical success of adoptive cell therapy with chimeric antigen receptor (CAR) T cells for treating hematological malignancies has revolutionized the field of cellular immunotherapy. However, due to the nature of utilizing autologous T cells, affordability and availability are major hurdles, in addition to scientific challenges relating to CAR‐T therapy optimization. Natural killer (NK) cell is a specialized immune effector cell type that recognizes and kills targets without human leukocyte antigen (HLA) restriction and prior sensitization. CAR‐NK cells do not cause graft vs host disease and can be obtained from unrelated donors as well as pluripotent stem cells (PSC), representing an ideal off‐the‐shelf therapeutics readily available for patients. Furthermore, unlike cytotoxic T cells, NK cells specifically target and eliminate cancer stem cells, which are the cells causing relapse and metastasis. PSCs can be genetically manipulated and engineered with CARs at the pluripotent stage, which allows the establishment of permanent, stable, and clonal PSC‐CAR lines for the manufacture of unlimited homogenous CAR‐NK cells. Multiple master PSC‐CAR cell banks targeting a variety of antigens for cancer, viral infection, and autoimmune diseases provide inexhaustible cell sources for all patients. Development of a next‐generation 3D bioreactor platform for PSC expansion and NK cell production overcomes major barriers related to cost and scalability for CAR‐NK product., The establishment of a master pluripotent stem cell (PSC)‐CAR cell bank targeting a variety of specific antigens will provide inexhaustible cell sources for the manufacture of truly off‐the‐shelf CAR‐NK cells for all patients of cancer, infectious, and autoimmune diseases.

Nicotinamide, the amide form of Vitamin B3, is a common nutrient supplement that plays important role in human fetal development. Nicotinamide has been widely used in clinical treatments, including the treatment of diseases during pregnancy. However, its impacts during embryogenesis have not been fully understood. In this study, we show that nicotinamide plays multiplex roles in mesoderm differentiation of human embryonic stem cells (hESCs). Nicotinamide promotes cardiomyocyte fate from mesoderm progenitor cells, and suppresses the emergence of other cell types. Independent of its functions in PARP and Sirtuin pathways, nicotinamide modulates differentiation through kinase inhibition. A KINOMEscan assay identifies 14 novel nicotinamide targets among 468 kinase candidates. We demonstrate that nicotinamide promotes cardiomyocyte differentiation through p38 MAP kinase inhibition. Furthermore, we show that nicotinamide enhances cardiomyocyte survival as a Rho-associated protein kinase (ROCK) inhibitor. This study reveals nicotinamide as a pleiotropic molecule that promotes the derivation and survival of cardiomyocytes, and it could become a useful tool for cardiomyocyte production for regenerative medicine. It also provides a theoretical foundation for physicians when nicotinamide is considered for treatments for pregnant women.

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation. [BMB Reports 2021; 54(10): 505-515].

Summary Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (α-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg Syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of α-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell–derived myotubes. FKRP-deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell–derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. In addition, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis., Graphical abstract, Highlights • The lysosome pathway is deregulated in FKRP-deficient myotubes • Autophagy is decreased in patient-specific LGMDR9 and WWS iPS cell–derived myotubes • FKRP WWS and LGMDR9 iPS cell–derived myotubes have increased apoptosis • FKRP correction in WWS myotubes rescues changes in autophagy and apoptosis, In this article, Perlingeiro and colleagues show that FKRP-deficient myotubes display transcriptome alterations in genes involved in extracellular matrix receptor interactions and lysosomal function. Using a panel of patient-specific LGMDR9 and WWS iPS cell–derived myotubes, they show a significant reduction in the autophagy-lysosome pathway and an increase in apoptosis, suggesting that these processes may contribute to the FKRP-associated MD pathogenesis.

Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future., 温度感受性センダイウイルスベクターを用いて ヒトES細胞／iPS細胞から骨格筋細胞を簡便に作製する技術開発 --神経筋疾患病態モデル構築と創薬研究への利用--. 京都大学プレスリリース. 2021-09-13.

The heart is the earliest organ to develop during embryogenesis and is remarkable in its ability to function efficiently as it is being sculpted. Cardiac heart defects account for a high burden of childhood developmental disorders with many remaining poorly understood mechanistically. Decades of work across a multitude of model organisms has informed our understanding of early cardiac differentiation and morphogenesis and has simultaneously opened new and unanswered questions. Here we have synthesized current knowledge in the field and reviewed recent developments in the realm of imaging, bioengineering and genetic technology and ex vivo cardiac modeling that may be deployed to generate more holistic models of early cardiac morphogenesis, and by extension, new platforms to study congenital heart defects.

Summary In Parkinson’s disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery., Highlights • Derivation of distinct dopaminergic subpopulations from pluripotent stem cells • Wnt signaling inhibitors promote SN dopaminergic neuron specification • Modeling selective vulnerability of SN dopaminergic neurons in vitro • Proteomics reveals pathways that promote SN dopaminergic neuron survival, In this article, Panman and colleagues show that by modulating Wnt signaling levels, distinct subpopulations of dopaminergic neurons can be derived from mouse and human pluripotent stem cells. Cultures enriched for substantia nigra dopaminergic neurons recapitulate the selective sensitivity to mitochondrial toxins as observed in Parkinson’s disease patients. The developed strategy facilitates disease modeling and drug discovery.

Summary The specification of inhibitory neurons has been described for the mouse and human brain, and many studies have shown that pluripotent stem cells (PSCs) can be used to create interneurons in vitro. It is unclear whether in vitro methods to produce human interneurons generate all the subtypes found in brain, and how similar in vitro and in vivo interneurons are. We applied single-nuclei and single-cell transcriptomics to model interneuron development from human cortex and interneurons derived from PSCs. We provide a direct comparison of various in vitro interneuron derivation methods to determine the homogeneity achieved. We find that PSC-derived interneurons capture stages of development prior to mid-gestation, and represent a minority of potential subtypes found in brain. Comparison with those found in fetal or adult brain highlighted decreased expression of synapse-related genes. These analyses highlight the potential to tailor the method of generation to drive formation of particular subtypes., Highlights • Comparison of interneurons derived from human pluripotent cells by various methods • Single-cell analyses define heterogeneity of in vitro-derived interneurons • Direct comparison of in vitro- and in vivo-derived interneurons • Identification of transcriptional modules that developmentally define interneurons, Plath, Lowry and colleagues profile interneurons generated from human pluripotent stem cells by various methods to understand the heterogeneity and cellular state of interneuron cultures in vitro. Using single-cell analyses, the authors define the homogeneity and maturity achieved with each in vitro method. By directly comparing these interneurons with those born in the human brain, the authors highlight distinctions particularly in synaptic genes and transcription factor modules that distinguish in vitro- and in vivo-derived neurons.

Summary For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies., Highlights • A Canadian resource (CFIT) has CF donor-matched iPSCs and nasal epithelial cells • Lung progenitor cells (LPCs) differentiated from iPSCs express CFTR • LPCs from people with rare CFTR mutations enable high-throughput therapy testing • Matching nasal cultures can validate patient-specific drug responses in LPCs, Bear and colleagues show that lung progenitor cells (LPCs) differentiated from cystic fibrosis (CF) iPSCs recapitulate the primary defects conferred by different types of CF mutations. LPCs enable high-throughput testing of interventions, and pilot studies show that responses in LPCs can be validated in patient-matched primary nasal epithelial cultures, confirming the potential utility of LPCs in precision CF therapy development.

Cardiomyocyte differentiation occurs through complex and finely regulated processes including cardiac lineage commitment and maturation from pluripotent stem cells (PSCs). To gain some insight into the genome-wide characteristics of cardiac lineage commitment, we performed transcriptome analysis on both mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs) at specific stages of cardiomyocyte differentiation. Specifically, the gene expression profiles and the protein-protein interaction networks of the mESC-derived plateletderived growth factor receptor-alpha (PDGFRα)+ cardiac lineagecommitted cells (CLCs) and hiPSC-derived kinase insert domain receptor (KDR)+ and PDGFRα+ cardiac progenitor cells (CPCs) at cardiac lineage commitment were compared with those of mesodermal cells and differentiated cardiomyocytes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the genes significantly upregulated at cardiac lineage commitment were associated with responses to organic substances and external stimuli, extracellular and myocardial contractile components, receptor binding, gated channel activity, PI3K‑AKT signaling, and cardiac hypertrophy and dilation pathways. Protein-protein interaction network analysis revealed that the expression levels of genes that regulate cardiac maturation, heart contraction, and calcium handling showed a consistent increase during cardiac differentiation; however, the expression levels of genes that regulate cell differentiation and multicellular organism development decreased at the cardiac maturation stage following lineage commitment. Additionally, we identified for the first time the protein-protein interaction network connecting cardiac development, the immune system, and metabolism during cardiac lineage commitment in both mESC-derived PDGFRα+ CLCs and hiPSC-derived KDR+PDGFRα+ CPCs. These findings shed light on the regulation of cardiac lineage commitment and the pathogenesis of cardiometabolic diseases. [BMB Reports 2021; 54(9): 464-469].

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4–5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90(+) cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p