Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated ...tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.
The polarity of microtubules is thought to be involved in spindle assembly, cytokinesis or active molecular transport. However, its exact role remains poorly understood, mainly because of the ...challenge to measure microtubule polarity in intact cells. We report here the use of fast Interferometric Second Harmonic Generation microscopy to study the polarity of microtubules forming the mitotic spindles in a zebrafish embryo. This technique provides a powerful tool to study mitotic spindle formation and may be directly transferable for investigating the kinetics and function of microtubule polarity in other aspects of subcellular motility or in native tissues.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Neutron star mergers offer unique conditions for the creation of the heavy elements, and additionally provide a testbed for our understanding of this synthesis known as the r-process. We have ...performed dynamical nucleosynthesis calculations and identified a single isotope, 254Cf, which has a particularly high impact on the brightness of electromagnetic transients associated with mergers on the order of 15 to 250 days. This is due to the anomalously long half-life of this isotope and the efficiency of fission thermalization compared to other nuclear channels. We estimate the fission fragment yield of this nucleus and outline the astrophysical conditions under which 254Cf has the greatest impact to the light curve. Future observations in the mid-infrared, which are bright during this regime, could indicate the production of actinide nucleosynthesis.
A recent study found a new, purely empirical correlation between two-neutron separation energies and neutron capture cross sections in keV neutron energy regimes. In shape/phase transition regimes, ...such as that near A= 150, S
2
n
values show an anomaly—a flattening of the normal near linear decrease with neutron number. This paper addresses two questions: (1) Using this new correlation, is this anomaly in S
2
n
values sizeable enough to produce an observable effect in capture cross sections? and (2) Can the correlation be used to quantitatively reproduce the cross sections in the transition region? It is found that the answer to both questions is in the affirmative. Possible relations to the
r
-process are briefly discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
RATIONALE:Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell–derived cardiomyocyte transplantation, thereby potentially preventing dilative ...remodeling and progression to heart failure.
OBJECTIVE:Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.
METHODS AND RESULTS:We constructed EHMs from human embryonic stem cell–derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, −6.7±1.4% versus control, −10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.
CONCLUSIONS:EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
Greater understanding of the spatial relationships between members of the human gut microbiota and available nutrients is needed to gain deeper insights about community dynamics and expressed ...functions. Therefore, we generated a panel of artificial food particles with each type composed of microscopic paramagnetic beads coated with a fluorescent barcode and one of 60 different dietary or host glycan preparations. Analysis of 160 Bacteroides and Parabacteroides strains disclosed diverse strain-specific and glycan-specific binding phenotypes. We identified carbohydrate structures that correlated with binding by specific bacterial strains in vitro and noted strain-specific differences in the catabolism of glycans that mediate adhesion. Mixed in vitro cultures revealed that these adhesion phenotypes are maintained in more complex communities. Additionally, orally administering glycan beads to gnotobiotic mice confirmed specificity in glycan binding. This approach should facilitate analyses of how strains occupying the same physical niche interact, and it should advance the development of synbiotics, more nutritious foods, and microbiota-based diagnostics.
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•Library of glass beads with unique multi-fluorescent labels and glycans was created•Imaging and 16S rRNA in vitro assays assessed gut bacterial strain glycan binding•Strain-specific binding correlated with linkage composition of dietary glycans•Administering glycan beads to gnotobiotic mice confirms specificity of binding
Using fluorescently labeled, microscopic glass beads containing different bound glycans, Patnode et al. examine carbohydrate-dependent adhesion of gut bacterial strains. Strain-specific binding correlated with linkage composition of dietary glycans and adhesion was observed in mixed in vitro cultures and gnotobiotic mice. The method has basic scientific plus diagnostic and therapeutic applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. ...Although current differentiation protocols yield hPSC‐CMs to >90% efficiency, hPSC‐CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR‐90 line) were differentiated to hPSC‐derived cardiomyocytes (hPSC‐CMs) in vitro using a small molecule based protocol. hPSC‐CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC‐CMs were mixed with 0.4 × 106 human fibroblasts (IMR‐90 line) (3:1 ratio) and type‐I collagen. The blend was cast into custom‐made 12‐mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC‐derived EHMs are comparable with rat neonatal cardiomyocyte‐derived EHMs. Three‐dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin‐T, calcium and potassium ion channels, β‐adrenergic receptors, and t‐tubule protein caveolin‐3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale‐up productions for clinical use in cardiovascular tissue engineering. Stem Cells 2018;36:265–277
Passive stretch affects the structural and functional maturation of engineered heart muscles (EHMs). Based on predictive computational modeling, results show how to optimize gene expression, cell alignment, calcium dynamics, and force generation of EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale‐up production for clinical use in cardiovascular tissue engineering.
High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses ...that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non-tumor-bearing and orthotopic glioma-bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.
To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a ...human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×109 CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.
•We present a strategy to optimize cardiac differentiation in suspension for hiPSCs.•The matrix-free suspension platform integrates hPSC expansion and differentiation.•Cardiac production in suspension achieves >90% purity with 1L spinner flasks.•The production process in suspension is defined, scalable, and GMP compliant.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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