A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1.

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Induction of mouse totipotent stem cells by a defined chemical cocktail.

In mice, only the zygotes and blastomeres from 2-cell embryos are authentic totipotent stem cells (TotiSCs) capable of producing all the differentiated cells in both embryonic and extraembryonic tissues and forming an entire organism1. However, it remains unknown whether and how totipotent stem cells can be established in vitro in the absence of germline cells. Here we demonstrate the induction and long-term maintenance of TotiSCs from mouse pluripotent stem cells using a combination of three small molecules: the retinoic acid analogue TTNPB, 1-azakenpaullone and the kinase blocker WS6. The resulting chemically induced totipotent stem cells (ciTotiSCs), resembled mouse totipotent 2-cell embryo cells at the transcriptome, epigenome and metabolome levels. In addition, ciTotiSCs exhibited bidirectional developmental potentials and were able to produce both embryonic and extraembryonic cells in vitro and in teratoma. Furthermore, following injection into 8-cell embryos, ciTotiSCs contributed to both embryonic and extraembryonic lineages with high efficiency. Our chemical approach to totipotent stem cell induction and maintenance provides a defined in vitro system for manipulating and developing understanding of the totipotent state and the development of multicellular organisms from non-germline cells.

Modified Shenlingbaizhu Decoction represses the pluripotency of colorectal cancer stem cells by inhibiting TGF-ß mediated EMT program.

BACKGROUND: The Modified Shenlingbaizhu Decoction (MSD) utilizes various phytomedicines has been applied to treat colorectal cancer (CRC). Colorectal cancer stem cells (CSCs) have proven to be tightly associated with CRC progression and metastasis. The mechanism of MSD's inhibitory effect on CSCs has not been determined. PURPOSE: To figure out how MSD inhibits the pluripotency of CSCs and impedes the EMT program. METHODS: The ingredients of MSD extracts were characterized by high-performance liquid chromatography (HPLC). BALB/c-nu mice were transplanted into EGFP labeled SW480 CRC cells and the tumor weight and volume were recorded before and after various doses of MSD treatment. The concentration of TGF-ß1 was quantified with an Enzyme-linked immunosorbent assay. To delineate the logical relationship between EMT and CSCs regulated by MSD, TGF-ß/Smad inhibitor and activator were adopted in tumor-bearing mice and diverse CRC cell lines. Cancer stem cell markers were analyzed by flow cytometry. In vitro analysis of cell motility and viability were done using CCK-8, wound healing, and invasion assay. Immunohistochemistry (IHC) and western blotting (WB) were used for detecting protein expression. The collected results were statistically analyzed with GraphPad Prism 8.0. RESULTS: MSD treatment significantly reduced the size of colorectal cancer tumors and lowered the serum content of TGF-ß1 in mice. Importantly, MSD markedly reduced the expression of pluripotent factors and depressed CD133+ stem cells in the tumor tissues. The TGF-ß/Smad inhibitor neutralized the EMT signaling and lowered the pluripotency by dephosphorylation of SMAD2/3. Similarly, MSD attenuated the pluripotency by limiting TGF-ß/Smad signaling-induced EMT in vivo. MSD inhibited colorectal cancer cell proliferation, migration, and invasion. CONCLUSIONS: MSD inhibits the growth of colorectal cancer. It dampens the pluripotency of CSCs by repressing the TGF-ß-induced EMT program.

Mitochondrial-Targeted Therapy for Doxorubicin-Induced Cardiotoxicity.

Anthracyclines, such as doxorubicin, are effective chemotherapeutic agents for the treatment of cancer, but their clinical use is associated with severe and potentially life-threatening cardiotoxicity. Despite decades of research, treatment options remain limited. The mitochondria is commonly considered to be the main target of doxorubicin and mitochondrial dysfunction is the hallmark of doxorubicin-induced cardiotoxicity. Here, we review the pathogenic mechanisms of doxorubicin-induced cardiotoxicity and present an update on cardioprotective strategies for this disorder. Specifically, we focus on strategies that can protect the mitochondria and cover different therapeutic modalities encompassing small molecules, post-transcriptional regulators, and mitochondrial transfer. We also discuss the shortcomings of existing models of doxorubicin-induced cardiotoxicity and explore advances in the use of human pluripotent stem cell derived cardiomyocytes as a platform to facilitate the identification of novel treatments against this disorder.

Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

Molecular study of the proliferation process of beta cells derived from pluripotent stem cells.

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.

Leveraging the Genetic Diversity of Human Stem Cells in Therapeutic Approaches.

Since their discovery 15 years ago, human pluripotent stem cell (hPSC) technologies have begun to revolutionize science and medicine, rapidly expanding beyond investigative research to drug discovery and development. Efforts to leverage hPSCs over the last decade have focused on increasing both the complexity and in vivo fidelity of human cellular models through enhanced differentiation methods. While these evolutions have fostered novel insights into disease mechanisms and influenced clinical drug discovery and development, there are still several considerations that limit the utility of hPSC models. In this review, we highlight important, yet underexplored avenues to broaden their reach. We focus on (i) the importance of diversifying existing hPSC collections, and their utilization to investigate therapeutic strategies in individuals from different genetic backgrounds, ancestry and sex; (ii) considerations for the selection of therapeutically relevant hPSC-based models; (iii) strategies to adequately increase the scale of cell-based studies; and (iv) the advances and constraints of clinical trials in a dish. Moreover, we advocate for harnessing the translational capabilities of hPSC models along with the use of innovative, scalable approaches for understanding genetic biases and the impact of sex and ancestry on disease mechanisms and drug efficacy and response. The next decade of hPSC innovation is poised to provide vast insights into the genetic basis of human disease and enable rapid advances to develop, repurpose, and ensure the safety of the next generation of disease therapies across diverse human populations.

Development of Functional Thyroid C Cell-like Cells from Human Pluripotent Cells in 2D and in 3D Scaffolds.

Medullary thyroid carcinoma contributes to about 3-4% of thyroid cancers and affects C cells rather than follicular cells. Thyroid C cell differentiation from human pluripotent stem cells has not been reported. We report the stepwise differentiation of human embryonic stem cells into thyroid C cell-like cells through definitive endoderm and anterior foregut endoderm and ultimobranchial body-like intermediates in monolayer and 3D Matrigel culture conditions. The protocol involved sequential treatment with interferon/transferrin/selenium/pyruvate, foetal bovine serum, and activin A, then IGF-1 (Insulin-like growth factor 1), on the basis of embryonic thyroid developmental sequence. As well as expressing C cell lineage relative to follicular-lineage markers by qPCR (quantitative polymerase chain reaction) and immunolabelling, these cells by ELISA (enzyme-linked immunoassay) exhibited functional properties in vitro of calcitonin storage and release of calcitonin on calcium challenge. This method will contribute to developmental studies of the human thyroid gland and facilitate in vitro modelling of medullary thyroid carcinoma and provide a valuable platform for drug screening.

Generation of heart-forming organoids from human pluripotent stem cells.

Heart-forming organoids (HFOs) derived from human pluripotent stem cells (hPSCs) are a complex, highly structured in vitro model of early heart, foregut and vasculature development. The model represents a potent tool for various applications, including teratogenicity studies, gene function analysis and drug discovery. Here, we provide a detailed protocol describing how to form HFOs within 14 d. In an initial 4 d preculture period, hPSC aggregates are individually formed in a 96-well format and then Matrigel-embedded. Subsequently, the chemical WNT pathway modulators CHIR99021 and IWP2 are applied, inducing directed differentiation. This highly robust protocol can be used on many different hPSC lines and be combined with manipulation technologies such as gene targeting and drug testing. HFO formation can be assessed by numerous complementary methods, ranging from various imaging approaches to gene expression studies. Here, we highlight the flow cytometry-based analysis of individual HFOs, enabling the quantitative monitoring of lineage formation.

Neurogenic Potential of the 18-kDa Mitochondrial Translocator Protein (TSPO) in Pluripotent P19 Stem Cells.

The 18-kDa translocator protein (TSPO) is a key mitochondrial target by which different TSPO ligands exert neuroprotective effects. We assayed the neurogenic potential of TSPO to induce the neuronal differentiation of pluripotent P19 stem cells in vitro. We studied changes in cell morphology, cell proliferation, cell death, the cell cycle, mitochondrial functionality, and the levels of pluripotency and neurogenesis of P19 stem cells treated with the TSPO ligand, PK 11195, in comparison to differentiation induced by retinoid acid (RA) and undifferentiated P19 stem cells. We observed that PK 11195 was able to activate the differentiation of P19 stem cells by promoting the development of embryoid bodies. PK 11195 also induced changes in the cell cycle, decreased cell proliferation, and activated cell death. Mitochondrial metabolism was also enhanced by PK 11195, thus increasing the levels of reactive oxygen species, Ca2+, and ATP as well as the mitochondrial membrane potential. Markers of pluripotency and neurogenesis were also altered during the cell differentiation process, as PK 11195 induced the differentiation of P19 stem cells with a high predisposition toward a neuronal linage, compared to cell differentiation induced by RA. Thus, we suggest a relevant neurogenic potential of TSPO along with broad therapeutic implications.

Nitro-Oleic Acid Inhibits Stemness Maintenance and Enhances Neural Differentiation of Mouse Embryonic Stem Cells via STAT3 Signaling.

Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and ß-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.

Activation of HIF1α Rescues the Hypoxic Response and Reverses Metabolic Dysfunction in the Diabetic Heart.

Type 2 diabetes (T2D) impairs hypoxia-inducible factor (HIF)1α activation, a master transcription factor that drives cellular adaptation to hypoxia. Reduced activation of HIF1α contributes to the impaired post-ischemic remodeling observed following myocardial infarction in T2D. Molidustat is an HIF stabilizer currently undergoing clinical trials for the treatment of renal anemia associated with chronic kidney disease; however, it may provide a route to pharmacologically activate HIF1α in the T2D heart. In human cardiomyocytes, molidustat stabilized HIF1α and downstream HIF target genes, promoting anaerobic glucose metabolism. In hypoxia, insulin resistance blunted HIF1α activation and downstream signaling, but this was reversed by molidustat. In T2D rats, oral treatment with molidustat rescued the cardiac metabolic dysfunction caused by T2D, promoting glucose metabolism and mitochondrial function, while suppressing fatty acid oxidation and lipid accumulation. This resulted in beneficial effects on post-ischemic cardiac function, with the impaired contractile recovery in T2D heart reversed by molidustat treatment. In conclusion, pharmacological HIF1α stabilization can overcome the blunted hypoxic response induced by insulin resistance. In vivo this corrected the abnormal metabolic phenotype and impaired post-ischemic recovery of the diabetic heart. Therefore, molidustat may be an effective compound to further explore the clinical translatability of HIF1α activation in the diabetic heart.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

OBJECTIVES: For clinical applications of cell-based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier-free suspension culture shows potential for large-scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension. MATERIALS AND METHODS: hPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real-time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing. RESULTS: Our results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size-controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA-seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism-related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion. CONCLUSIONS: Our results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction.

AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells. METHODS: We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers. RESULTS: The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS+/C109Y patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response. CONCLUSIONS/INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.

Advancing models of neural development with biomaterials.

Human pluripotent stem cells have emerged as a promising in vitro model system for studying the brain. Two-dimensional and three-dimensional cell culture paradigms have provided valuable insights into the pathogenesis of neuropsychiatric disorders, but they remain limited in their capacity to model certain features of human neural development. Specifically, current models do not efficiently incorporate extracellular matrix-derived biochemical and biophysical cues, facilitate multicellular spatio-temporal patterning, or achieve advanced functional maturation. Engineered biomaterials have the capacity to create increasingly biomimetic neural microenvironments, yet further refinement is needed before these approaches are widely implemented. This Review therefore highlights how continued progression and increased integration of engineered biomaterials may be well poised to address intractable challenges in recapitulating human neural development.

Global Transcriptional Analyses of the Wnt-Induced Development of Neural Stem Cells from Human Pluripotent Stem Cells.

The differentiation of human pluripotent stem cells (hPSCs) to neural stem cells (NSCs) is the key initial event in neurogenesis and is thought to be dependent on the family of Wnt growth factors, their receptors and signaling proteins. The delineation of the transcriptional pathways that mediate Wnt-induced hPSCs to NSCs differentiation is vital for understanding the global genomic mechanisms of the development of NSCs and, potentially, the creation of new protocols in regenerative medicine. To understand the genomic mechanism of Wnt signaling during NSCs development, we treated hPSCs with Wnt activator (CHIR-99021) and leukemia inhibitory factor (LIF) in a chemically defined medium (N2B27) to induce NSCs, referred to as CLNSCs. The CLNSCs were subcultured for more than 40 passages in vitro; were positive for AP staining; expressed neural progenitor markers such as NESTIN, PAX6, SOX2, and SOX1; and were able to differentiate into three neural lineage cells: neurons, astrocytes, and oligodendrocytes in vitro. Our transcriptome analyses revealed that the Wnt and Hedgehog signaling pathways regulate hPSCs cell fate decisions for neural lineages and maintain the self-renewal of CLNSCs. One interesting network could be the deregulation of the Wnt/ß-catenin signaling pathway in CLNSCs via the downregulation of c-MYC, which may promote exit from pluripotency and neural differentiation. The Wnt-induced spinal markers HOXA1-4, HOXA7, HOXB1-4, and HOXC4 were increased, however, the brain markers FOXG1 and OTX2, were absent in the CLNSCs, indicating that CLNSCs have partial spinal cord properties. Finally, a CLNSC simple culture condition, when applied to hPSCs, supports the generation of NSCs, and provides a new and efficient cell model with which to untangle the mechanisms during neurogenesis.

Chalcone-Supported Cardiac Mesoderm Induction in Human Pluripotent Stem Cells for Heart Muscle Engineering.

Human pluripotent stem cells (hPSCs) hold great promise for applications in cell therapy and drug screening in the cardiovascular field. Bone morphogenetic protein 4 (BMP4) is key for early cardiac mesoderm induction in hPSC and subsequent cardiomyocyte derivation. Small-molecular BMP4 mimetics may help to standardize cardiomyocyte derivation from hPSCs. Based on observations that chalcones can stimulate BMP4 signaling pathways, we hypothesized their utility in cardiac mesoderm induction. To test this, we set up a two-tiered screening strategy, (1) for directed differentiation of hPSCs with commercially available chalcones (4'-hydroxychalcone [4'HC] and Isoliquiritigen) and 24 newly synthesized chalcone derivatives, and (2) a functional screen to assess the propensity of the obtained cardiomyocytes to self-organize into contractile engineered human myocardium (EHM). We identified 4'HC, 4-fluoro-4'-methoxychalcone, and 4-fluoro-4'-hydroxychalcone as similarly effective in cardiac mesoderm induction, but only 4'HC as an effective replacement for BMP4 in the derivation of contractile EHM-forming cardiomyocytes.

Emerging Opportunities in Human Pluripotent Stem-Cells Based Assays to Explore the Diversity of Botulinum Neurotoxins as Future Therapeutics.

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and are responsible for botulism, a fatal disorder of the nervous system mostly induced by food poisoning. Despite being one of the most potent families of poisonous substances, BoNTs are used for both aesthetic and therapeutic indications from cosmetic reduction of wrinkles to treatment of movement disorders. The increasing understanding of the biology of BoNTs and the availability of distinct toxin serotypes and subtypes offer the prospect of expanding the range of indications for these toxins. Engineering of BoNTs is considered to provide a new avenue for improving safety and clinical benefit from these neurotoxins. Robust, high-throughput, and cost-effective assays for BoNTs activity, yet highly relevant to the human physiology, have become indispensable for a successful translation of engineered BoNTs to the clinic. This review presents an emerging family of cell-based assays that take advantage of newly developed human pluripotent stem cells and neuronal function analyses technologies.