ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Synthesis of Substituted Pyridines via Formal (3+3) Cycloaddition of Enamines with Unsaturated Aldehydes and Ketones.

An organocatalyzed, formal (3+3) cycloaddition reaction is described for the practical synthesis of substituted pyridines. Starting from readily available enamines and enal/ynal/enone substrates, the protocol affords tri- or tetrasubstituted pyridine scaffolds bearing various functional groups. This method was demonstrated on a 50 g scale, enabling the synthesis of 2-isopropyl-4-methylpyridin-3-amine, a raw material used for the manufacture of sotorasib. Mechanistic analysis using two-dimensional nuclear magnetic resonance (NMR) spectrometry revealed the transformation proceeds through the reversible formation of a stable reaction off-cycle species that precedes pyridine formation. In situ reaction progress kinetic analysis and control NMR studies were employed to better understand the role of FeCl3 and pyrrolidine hydrochloride in promoting the reaction.

Sustainability Challenges in Peptide Synthesis and Purification: From R&D to Production.

In recent years, there has been a growing interest in therapeutic peptides within the pharmaceutical industry with more than 50 peptide drugs on the market, approximately 170 in clinical trials, and >200 in preclinical development. However, the current state of the art in peptide synthesis involves primarily legacy technologies with use of large amounts of highly hazardous reagents and solvents and little focus on green chemistry and engineering. In 2016, the ACS Green Chemistry Institute Pharmaceutical Roundtable identified development of greener processes for peptide API as a critical unmet need, and as a result, a new Roundtable team formed to address this important area. The initial focus of this new team is to highlight best practices in peptide synthesis and encourage much needed innovations. In this Perspective, we aim to summarize the current challenges of peptide synthesis and purification in terms of sustainability, highlight possible solutions, and encourage synergies between academia, the pharmaceutical industry, and contract research organizations/contract manufacturing organizations.

Development of a Commercial Process To Prepare AMG 232 Using a Green Ozonolysis-Pinnick Tandem Transformation.

A robust process to manufacture AMG 232 was developed to deliver drug substance of high purity. Highlights of the commercial process development efforts include the following: (i) use of a novel bench-stable Vilsmeier reagent, methoxymethylene- N, N-dimethyliminium methyl sulfate, for selective in situ activation of a primary alcohol intermediate; (ii) use of a new crystalline and stable isopropyl calcium sulfinate reagent ensuring robust preparation of a sulfone intermediate; (iii) development of a safe ozonolysis process conducted in an aqueous solvent mixture in either batch or continuous manufacturing mode; and (iv) control of the drug substance purity by crystallization of a salt rejecting impurities effectively. The new process was demonstrated to afford the drug substance (99.9 LC area %) in 49.8% overall yield from starting material DLAC (1).

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.

Transcription factor Stat3 directs self-renewal of pluripotent mouse embryonic stem (ES) cells downstream of the cytokine leukemia inhibitory factor (LIF). Stat3 upregulates pivotal transcription factors in the ES cell gene regulatory network to sustain naïve identity. Stat3 also contributes to the rapid proliferation of ES cells. Here, we show that Stat3 increases the expression of mitochondrial-encoded transcripts and enhances oxidative metabolism. Chromatin immunoprecipitation reveals that Stat3 binds to the mitochondrial genome, consistent with direct transcriptional regulation. An engineered form of Stat3 that localizes predominantly to mitochondria is sufficient to support enhanced proliferation of ES cells, but not to maintain their undifferentiated phenotype. Furthermore, during reprogramming from primed to naïve states of pluripotency, Stat3 similarly upregulates mitochondrial transcripts and facilitates metabolic resetting. These findings suggest that the potent stimulation of naïve pluripotency by LIF/Stat3 is attributable to parallel and synergistic induction of both mitochondrial respiration and nuclear transcription factors.

Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.

The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.

Rhodium-catalyzed enantioselective vinylogous addition of enol ethers to vinyldiazoacetates.

A highly asymmetric vinylogous addition of acyclic silyl enol ethers to siloxyvinyldiazoacetate is described. The reaction features a diastereoselective 1,4-siloxy group migration event. Products are obtained in up to 97% ee. When more sterically crowded silyl enol ethers are employed, an enantioselective formal [3+2] cycloaddition becomes the dominant reaction pathway. Control experiments reveal the (Z)-olefin geometry to be critical for high levels of enantiocontrol.

Polarity inversion of donor-acceptor cyclopropanes: disubstituted δ-lactones via enantioselective iridium catalysis.

The coupling of carbonyl electrophiles at the donor position of donor-acceptor cyclopropanes is described, representing an inversion of polarity with respect to conventional reactivity modes displayed by these reagents. Specifically, upon exposure of donor-acceptor cyclopropanes to alcohols in the presence of a cyclometalated iridium catalyst modified by (S)-BINAP, catalytic C-C coupling occurs, providing enantiomerically enriched products of carbonyl allylation. Identical products are obtained upon isopropanol-mediated transfer hydrogenation of donor-acceptor cyclopropanes in the presence of aldehydes. The reaction products are directly transformed to cis-4,5-disubstituted δ-lactones.

Cyclopropane-aldehyde annulations at quaternary donor sites: stereoselective access to highly substituted tetrahydrofurans.

A diastereoselective synthesis of pentasubstituted tetrahydrofurans via a Lewis acid catalyzed (3 + 2)-annulation of quaternary donor site cyclopropanes and aldehydes is described. The reaction is catalyzed by Sn(OTf)(2), SnCl(4), or Hf(OTf)(4) in yields up to 95% and diastereomeric ratios as high as 99:1.

The ground state of pluripotency.

Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.

Imaging-based chemical screens using normal and glioma-derived neural stem cells.

The development of optimal culture methods for embryonic, tissue and cancer stem cells is a critical foundation for their application in drug screening. We previously described defined adherent culture conditions that enable expansion of human radial glia-like fetal NS (neural stem) cells as stable cell lines. Similar protocols proved effective in the establishment of tumour-initiating stem cell lines from the human brain tumour glioblastoma multiforme, which we termed GNS (glioma NS) cells. Others have also recently derived more primitive human NS cell lines with greater neuronal subtype differentiation potential than NS cells, which have similarities to the early neuroepithelium, named NES (neuroepithelial stem) cells. In the present paper, we discuss the utility of these cells for chemical screening, and describe methods for a simple high-content live-image-based platform. We report the effects of a panel of 160 kinase inhibitors (Inhibitor Select I and II; Calbiochem) on NES cells, identifying three inhibitors of ROCK (Rho-associated kinase) as promoting the expansion of NES cell cultures. For the GNS cells, we screened a panel of 1000 compounds and confirmed our previous finding of a cytotoxic effect of modulators of neurotransmitter signalling pathways. These studies provide a framework for future higher-throughput screens.

Dynamic kinetic asymmetric synthesis of substituted pyrrolidines from racemic cyclopropanes and aldimines: reaction development and mechanistic insights.

An enantioselective preparation of 2,5-cis-disubstituted pyrrolidines has been achieved via a dynamic kinetic asymmetric transformation (DyKAT) of racemic donor-acceptor cyclopropanes and (E)-aldimines. Mechanistic studies suggest that isomerization of the aldimine or resultant iminium to the Z geometry is not a pathway that furnishes the observed 2,5-cis-disubstituted products.

Lewis acid-promoted Friedel-Crafts alkylation reactions with alpha-ketophosphate electrophiles.

The BF(3).OEt(2)-promoted nucleophilic substitution of alpha-aryl-alpha-ketophosphates to afford alpha,alpha-diaryl ketone products is described. Electron-rich alpha-ketophosphates perform best, with electron-neutral and electron-poor substrates also tolerated. The reaction is tolerant of a range of aromatic, heteroaromatic, and nonaromatic nucleophiles, with yields ranging from 44% to 84%. Enantioenriched starting material yields racemic product, suggesting an S(N)1 pathway via an acylcarbenium ion.

Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.

Neuroepithelial cells supply an initial transient wave of MSC differentiation.

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.

Notch promotes neural lineage entry by pluripotent embryonic stem cells.

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.

Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors.

Reliable in vitro conversion of pluripotent embryonic stem (ES) cells into bone and cartilage-forming cells would expand opportunities for experimental investigations of skeletogenesis and could also provide new cellular sources for pharmaceutical screening and for cell therapy applications. Here, we evaluate the generation of mesenchymal cell lineages from mouse ES cells following treatment of embryoid bodies with retinoic acid, previously reported to induce development of adipocyte precursors. We find that retinoic acid reduces mesodermal differentiation but enhances expression of markers of neural crest, an alternative origin of mesenchymal elements. Runx1 and Ptprv appear to provide early markers of mesenchymal potential. Subsequently, different mesenchymal fates are generated in response to particular growth factors. Substitution of the adipogenic factors insulin and triiodothyronine with bone morphogenetic protein (BMP-4) results in suppression of adipogenesis and development of a mature osteogenic phenotype. In contrast, treatment with transforming growth factor-beta (TGF-beta3) promotes chondrogenic differentiation. Thus, the use of appropriate growth factors and culture milieu steers differentiation of ES cell-derived precursors into distinct mesenchymal compartments.

SoxB transcription factors specify neuroectodermal lineage choice in ES cells.

Knowledge of lineage decision machinery in pluripotent embryonic stem (ES) cells may shed light on the process of germ layer segregation in the mammalian embryo and enable directed differentiation in vitro for biomedical applications. We have investigated the contribution of Class B1 Sox transcription factors to lineage choice during ES cell differentiation. We report that forced expression of Sox1 or Sox2 did not impair propagation of undifferentiated ES cells, but upon release from self-renewal promoted differentiation into neuroectoderm at the expense of mesoderm and endoderm. The efficient specification of a primary lineage by transcription factor manipulation provides a paradigm for instructing differentiation of ES cells for biopharmaceutical screening and cell therapy applications.

Defined conditions for neural commitment and differentiation.

The efficiency of monolayer differentiation establishes that commitment of ES cells to a neural fate needs neither multicellular aggregation nor extrinsic inducers. The entire process by which pluripotent ES cells acquire neural specification can be visualized and recorded at the level of individual colonies. Furthermore this simple culture system is amenable to cellular and molecular dissection, promising to yield new insights into the mechanism underlying neural determination in mammals and perhaps to deliver the goal of "directed" homogeneous differentiation of ES cells.