Expression of the transcription factor PU.1 induces the generation of microglia-like cells in human cortical organoids.

Microglia play a role in the emergence and preservation of a healthy brain microenvironment. Dysfunction of microglia has been associated with neurodevelopmental and neurodegenerative disorders. Investigating the function of human microglia in health and disease has been challenging due to the limited models of the human brain available. Here, we develop a method to generate functional microglia in human cortical organoids (hCOs) from human embryonic stem cells (hESCs). We apply this system to study the role of microglia during inflammation induced by amyloid-ß (Aß). The overexpression of the myeloid-specific transcription factor PU.1 generates microglia-like cells in hCOs, producing mhCOs (microglia-containing hCOs), that we engraft in the mouse brain. Single-cell transcriptomics reveals that mhCOs acquire a microglia cell cluster with an intact complement and chemokine system. Functionally, microglia in mhCOs protect parenchyma from cellular and molecular damage caused by Aß. Furthermore, in mhCOs, we observed reduced expression of Aß-induced expression of genes associated with apoptosis, ferroptosis, and Alzheimer's disease (AD) stage III. Finally, we assess the function of AD-associated genes highly expressed in microglia in response to Aß using pooled CRISPRi coupled with single-cell RNA sequencing in mhCOs. In summary, we provide a protocol to generate mhCOs that can be used in fundamental and translational studies as a model to investigate the role of microglia in neurodevelopmental and neurodegenerative disorders.

Human pluripotent stem cell-derived eosinophils reveal potent cytotoxicity against solid tumors.

Eosinophils are attractive innate immune cells to use to potentiate T cell antitumor efficacy because they are capable of infiltrating tumors at early stages and modulating the tumor microenvironment. However, the limited number of functional eosinophils caused by the scarcity and short life of primary eosinophils in peripheral blood has greatly impeded the development of eosinophil-based immunotherapy. In this study, we established an efficient chemically defined protocol to generate a large quantity of functional eosinophils from human pluripotent stem cells (hPSCs) with nearly 100% purity expressing eosinophil peroxidase. These hPSC-derived eosinophils transcriptionally resembled their primary counterpart. Moreover, hPSC-derived eosinophils showed competent tumor killing capacity in established solid tumors. Furthermore, the combination of hPSC-derived eosinophils with CAR-T cells exhibited potential synergistic effects, inhibiting tumor growth and enhancing mouse survival. Our study opens up new avenues for the development of eosinophil-based immunotherapies to treat cancer.

Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

Human embryoid bodies as a 3D tissue model of the extracellular matrix and α-dystroglycanopathies.

The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies and specific types of brain malformation. The latter three pathologies occur in the α-dystroglycanopathies, which are caused by dysfunction of the ECM receptor α-dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In α-dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the α-dystroglycanopathies. Finally, we apply this system to evaluate an experimental ribitol supplement therapy on genetically diverse α-dystroglycanopathy patient samples.This article has an associated First Person interview with the first author of the paper.

Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis.

Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.

Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells.

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model.

MERTK-Dependent Ensheathment of Photoreceptor Outer Segments by Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

Maintenance of a healthy photoreceptor-retinal pigment epithelium (RPE) interface is essential for vision. At the center of this interface, apical membrane protrusions stemming from the RPE ensheath photoreceptor outer segments (POS), and are possibly involved in the recycling of POS through phagocytosis. The molecules that regulate POS ensheathment and its relationship to phagocytosis remain to be deciphered. By means of ultrastructural analysis, we revealed that Mer receptor tyrosine kinase (MERTK) ligands, GAS6 and PROS1, rather than αVß5 integrin receptor ligands, triggered POS ensheathment by human embryonic stem cell (hESC)-derived RPE. Furthermore, we found that ensheathment is required for POS fragmentation before internalization. Consistently, POS ensheathment, fragmentation, and internalization were abolished in MERTK mutant RPE, and rescue of MERTK expression in retinitis pigmentosa (RP38) patient RPE counteracted these defects. Our results suggest that loss of ensheathment due to MERTK dysfunction might contribute to vision impairment in RP38 patients.

Quantification of the morphological characteristics of hESC colonies.

The maintenance of the undifferentiated state in human embryonic stem cells (hESCs) is critical for further application in regenerative medicine, drug testing and studies of fundamental biology. Currently, the selection of the best quality cells and colonies for propagation is typically performed by eye, in terms of the displayed morphological features, such as prominent/abundant nucleoli and a colony with a tightly packed appearance and a well-defined edge. Using image analysis and computational tools, we precisely quantify these properties using phase-contrast images of hESC colonies of different sizes (0.1-1.1 [Formula: see text]) during days 2, 3 and 4 after plating. Our analyses reveal noticeable differences in their structure influenced directly by the colony area [Formula: see text]. Large colonies (A > 0.6 mm2) have cells with smaller nuclei and a short intercellular distance when compared with small colonies (A < 0.2 mm2). The gaps between the cells, which are present in small and medium sized colonies with A ≤ 0.6 mm2, disappear in large colonies (A > 0.6 mm2) due to the proliferation of the cells in the bulk. This increases the colony density and the number of nearest neighbours. We also detect the self-organisation of cells in the colonies where newly divided (smallest) cells cluster together in patches, separated from larger cells at the final stages of the cell cycle. This might influence directly cell-to-cell interactions and the community effects within the colonies since the segregation induced by size differences allows the interchange of neighbours as the cells proliferate and the colony grows. Our findings are relevant to efforts to determine the quality of hESC colonies and establish colony characteristics database.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Quantitative Proteomics Study Reveals Changes in the Molecular Landscape of Human Embryonic Stem Cells with Impaired Stem Cell Differentiation upon Exposure to Titanium Dioxide Nanoparticles.

The increasing number of nanoparticles (NPs) being used in various industries has led to growing concerns of potential hazards that NP exposure can incur on human health. However, its global effects on humans and the underlying mechanisms are not systemically studied. Human embryonic stem cells (hESCs), with the ability to differentiate to any cell types, provide a unique system to assess cellular, developmental, and functional toxicity in vitro within a single system highly relevant to human physiology. Here, the quantitative proteomics approach is adopted to evaluate the molecular consequences of titanium dioxide NPs (TiO2 NPs) exposure in hESCs. The study identifies ≈328 unique proteins significantly affected by TiO2 NPs exposure. Proteomics analysis highlights that TiO2 NPs can induce DNA damage, elevated oxidative stress, apoptotic responses, and cellular differentiation. Furthermore, in vivo analysis demonstrates remarkable reduction in the ability of hESCs in teratoma formation after TiO2 NPs exposure, suggesting impaired pluripotency. Subsequently, it is found that TiO2 NPs can disrupt hESC mesoderm differentiation into cardiomyocytes. The study unveils comprehensive changes in the molecular landscape of hESCs by TiO2 NPs and identifies the impact which TiO2 NPs can have on the pluripotency and differentiation properties of human stem cells.

Contribution of transposable elements and distal enhancers to evolution of human-specific features of interphase chromatin architecture in embryonic stem cells.

Transposable elements have made major evolutionary impacts on creation of primate-specific and human-specific genomic regulatory loci and species-specific genomic regulatory networks (GRNs). Molecular and genetic definitions of human-specific changes to GRNs contributing to development of unique to human phenotypes remain a highly significant challenge. Genome-wide proximity placement analysis of diverse families of human-specific genomic regulatory loci (HSGRL) identified topologically associating domains (TADs) that are significantly enriched for HSGRL and designated rapidly evolving in human TADs. Here, the analysis of HSGRL, hESC-enriched enhancers, super-enhancers (SEs), and specific sub-TAD structures termed super-enhancer domains (SEDs) has been performed. In the hESC genome, 331 of 504 (66%) of SED-harboring TADs contain HSGRL and 68% of SEDs co-localize with HSGRL, suggesting that emergence of HSGRL may have rewired SED-associated GRNs within specific TADs by inserting novel and/or erasing existing non-coding regulatory sequences. Consequently, markedly distinct features of the principal regulatory structures of interphase chromatin evolved in the hESC genome compared to mouse: the SED quantity is 3-fold higher and the median SED size is significantly larger. Concomitantly, the overall TAD quantity is increased by 42% while the median TAD size is significantly decreased (p = 9.11E-37) in the hESC genome. Present analyses illustrate a putative global role for transposable elements and HSGRL in shaping the human-specific features of the interphase chromatin organization and functions, which are facilitated by accelerated creation of novel transcription factor binding sites and new enhancers driven by targeted placement of HSGRL at defined genomic coordinates. A trend toward the convergence of TAD and SED architectures of interphase chromatin in the hESC genome may reflect changes of 3D-folding patterns of linear chromatin fibers designed to enhance both regulatory complexity and functional precision of GRNs by creating predominantly a single gene (or a set of functionally linked genes) per regulatory domain structures. Collectively, present analyses reveal critical evolutionary contributions of transposable elements and distal enhancers to creation of thousands primate- and human-specific elements of a chromatin folding code, which defines the 3D context of interphase chromatin both restricting and facilitating biological functions of GRNs.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds.

Collagen vitrigels with low-fibril density enhance human embryonic stem cell-derived retinal pigment epithelial cell maturation.

Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC-RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60 nm and Young's modulus of 2.41 ± 0.13 MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC-RPE maturation and presents a potential bioengineered substratum for hESC-RPE for future preclinical and clinical applications.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing.

Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors.

Transplantation of rod photoreceptors, derived either from neonatal retinae or pluripotent stem cells (PSCs), can restore rod-mediated visual function in murine models of inherited blindness. However, humans depend more upon cone photoreceptors that are required for daylight, color, and high-acuity vision. Indeed, macular retinopathies involving loss of cones are leading causes of blindness. An essential step for developing stem cell-based therapies for maculopathies is the ability to generate transplantable human cones from renewable sources. Here, we report a modified 2D/3D protocol for generating hPSC-derived neural retinal vesicles with well-formed ONL-like structures containing cones and rods bearing inner segments and connecting cilia, nascent outer segments, and presynaptic structures. This differentiation system recapitulates human photoreceptor development, allowing the isolation and transplantation of a pure population of stage-matched cones. Purified human long/medium cones survive and become incorporated within the adult mouse retina, supporting the potential of photoreceptor transplantation for treating retinal degeneration.

Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis.

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis.

Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.

The Ultrastructural Signature of Human Embryonic Stem Cells.

The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc.

Hi-C-constrained physical models of human chromosomes recover functionally-related properties of genome organization.

Combining genome-wide structural models with phenomenological data is at the forefront of efforts to understand the organizational principles regulating the human genome. Here, we use chromosome-chromosome contact data as knowledge-based constraints for large-scale three-dimensional models of the human diploid genome. The resulting models remain minimally entangled and acquire several functional features that are observed in vivo and that were never used as input for the model. We find, for instance, that gene-rich, active regions are drawn towards the nuclear center, while gene poor and lamina associated domains are pushed to the periphery. These and other properties persist upon adding local contact constraints, suggesting their compatibility with non-local constraints for the genome organization. The results show that suitable combinations of data analysis and physical modelling can expose the unexpectedly rich functionally-related properties implicit in chromosome-chromosome contact data. Specific directions are suggested for further developments based on combining experimental data analysis and genomic structural modelling.

Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes.

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.