High-Throughput Two-Photon 3D Printing Enabled by Holographic Multi-Foci High-Speed Scanning.

The emerging two-photon polymerization (TPP) technique enables high-resolution printing of complex 3D structures, revolutionizing micro/nano additive manufacturing. Various fast scanning and parallel processing strategies have been proposed to promote its efficiency. However, obtaining large numbers of uniform focal spots for parallel high-speed scanning remains challenging, which hampers the realization of higher throughput. We report a TPP printing platform that combines galvanometric mirrors and liquid crystal on silicon spatial light modulator (LCoS-SLM). By setting the target light field at LCoS-SLM's diffraction center, sufficient energy is acquired to support simultaneous polymerization of over 400 foci. With fast scanning, the maximum printing speed achieves 1.49 × 108 voxels s-1, surpassing the existing scanning-based TPP methods while maintaining high printing resolution and flexibility. To demonstrate the processing capability, functional 3D microstructure arrays are rapidly fabricated and applied in micro-optics and micro-object manipulation. Our method may expand the prospects of TPP in large-scale micro/nanomanufacturing.

Generation of induced pluripotent stem cell lines from helper and cytotoxic T cells of healthy individuals.

T lymphocytes are the most abundant mononuclear blood cells and can serve as a source for generating induced pluripotent stem cells (iPSCs) for disease modeling or drug development. Here, we report the derivation of two iPSC lines from CD4+ helper T cells and CD8+ cytolytic T cells, respectively. The reprogramming was performed using Sendai virus encoding Klf-4, c-Myc, Oct-4 and Sox-2. Both iPSC lines displayed typical embryonic stem cell-like morphology and normal karyotype. Pluripotency was confirmed using immunocytochemistry methods and teratoma formation assay.

Flocculation and dewatering of the Kaolin slurry treated by single- and dual-polymer flocculants.

To mitigate the sudden increase in the production of waste engineering slurry, predominantly composed of Kaolinite, this study investigated the flocculation and dewatering of Kaolin slurry treated with single- and dual-polymer flocculants. The influence of the flocculant type and dosage, under single- and dual-dose conditions, on slurry's sedimentation and the filtration characteristics, were thoroughly discussed. The results reveal that the adsorption bridging of the polymeric flocculant, resulting from hydrogen bonds, exerts a more significant effect than electrical neutralization on forming a large floc. Under single-dose conditions, nonionic polyacrylamides (NPAMs) with the strongest adsorption bridging leads to biggest flocs and the maximum settling rate of 21.55 mm/s. Under the dual-dose conditions of polymeric aluminium chloride (PAC) and PAM, the size of the slurry's floc decreases with an increase in PAC dosage. Nevertheless, the filtration performance of the slurry improves, with the lowest SRF value of the flocculated slurry being 1.58 × 1011 m/kg as 3‰ PAC and 3‰ NPAM is dosed. The improvement is explained by the micro-pore distribution of sludge. According to Mercury intrusion porosimetry (MIP) test, the slurry treated with the optimal dosage of dual-polymer flocculant exhibits the greatest sludge pore size and connected porosity (with a maximum value of 20.99%). Furthermore, the study discusses and compares the flocculation mechanism of single- and dual-polymer flocculants. The obtained results provide guidance for selecting appropriate flocculants for dewatering inorganic slurries, using different dewatering methods, such as gravitational thickening or filter pressing.

Convergence of human pluripotent stem cell, organoid, and genome editing technologies.

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.

MiR-140-3p Ameliorates the Progression of Osteoarthritis via Targeting CXCR4.

Osteoarthritis is a common disease character with progressive destruction of cartilage. MicroRNA (miR)-140-3p was validated as a biomarker for osteoarthritis. However, the mechanism by which miRNA-140-3p regulates osteoarthritis remains unclear. Thus, this study aims to evaluate the potential function of miRNA-140-3p during the pathogenesis of osteoarthritis. MiRNA-140-3p expression in tissue and CHON-001 chondrocyte cells was determined with quantitative real time (qRT)-PCR. In vitro osteoarthritis model was established by treatment of the chondrocyte cells CHON-001 with interleukin (IL)-1ß. Cell proliferation and apoptosis were measured with cell counting kit-8 (CCK8) and Annexin V/propidium iodide (PI) apoptosis assay, respectively. Protein expressions were evaluated using Western blot. The target gene of miR-140-3p was predicted using Targetscan and miRDB. MiR-140-3p was downregulated in knee tissue from patients with osteoarthritis. IL-1ß inhibited the proliferation of CHON-001 cells via inducing apoptosis. In addition, IL-1ß significantly inhibited the expressions of collagen II and aggrecan and increased the level of matrix metalloproteinase (MMP)13. However, the effects of IL-1ß could be ameliorated by the addition of miR-140-3p mimics. Moreover, luciferase reporter assay demonstrated CXCR4 as a target gene of miR-140-3p. IL-1ß-induced upregulation of CXCR4 could be blocked by miR-140-3p mimics. Our study indicated that miR-140-3p could suppress the progression of osteoarthritis by directly targeting CXCR4. Therefore, miR-140-3p might serve as a potential therapeutic target for the treatment of osteoarthritis.

Study on Targeting Relationship Between miR-320b and FGD5-AS1 and Its Effect on Biological Function of Osteosarcoma Cells.

OBJECTIVE: To probe into the expression of FGD5-AS1 in osteosarcoma and its relationship with miR-320b. METHODS: The tissue and serum samples of 97 patients with osteosarcoma were collected, and the serum samples of 100 healthy subjects who concurrently underwent physical examination were selected as the control. FGD5-AS1 expression in tissues and serum was detected, and osteosarcoma cells were transfected to measure cell behaviors such as proliferation, invasion and apoptosis. RESULTS: FGD5-AS1 was highly expressed in osteosarcoma, and its elevated expression indicated poor survival of patients. Serum FGD5-AS1 was related to tumor size and clinical stage and could be used for the diagnosis of osteosarcoma. The study of osteosarcoma cell lines U2OS and SaOS-2 showed that after inhibiting FGD5-AS1, the viability and invasion capacity of osteosarcoma cells decreased statistically compared with the control group (CG), while the apoptosis ability could be improved by further regulating apoptotic proteins (P<0.05). Detection of EMT-related proteins identified that E-cadherin increased while N-cadherin decreased significantly after FGD5-AS1 inhibition (P<0.05). Correlation analysis revealed a negative correlation between miR-320b and FGD5-AS1 (r = -0.410, P<0.001). Overexpression of miR-320b significantly inhibited cell viability, invasion and EMT ability, and increased the apoptosis rate, while inhibiting miR-320b expression produced the opposite results. The targeting relationship between miR-320b and FGD5-AS1 was confirmed through the biological prediction website, luciferase assay and RNA binding protein immunoprecipitation (RIP) assay. Inhibition of miR-320b could reverse the regulatory effect of FGD5-AS1 knockdown on osteosarcoma cells. CONCLUSION: FGD5-AS1 is highly expressed in osteosarcoma and is involved in the biological procession of osteosarcoma by targeting miR-320b.

Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing.

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells.

IMPACT STATEMENT: In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.

Transient c-Src Suppression During Endodermal Commitment of Human Induced Pluripotent Stem Cells Results in Abnormal Profibrotic Cholangiocyte-Like Cells.

Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317.

Biliary Atresia Relevant Human Induced Pluripotent Stem Cells Recapitulate Key Disease Features in a Dish.

Biliary atresia (BA) is the most common cause of pediatric end-stage liver disease and the etiology is poorly understood. There is no effective therapy for BA partly due to lack of human BA models. Towards developing in vitro human models of BA, disease-specific induced pluripotent stem cells (iPSCs) from 6 BA patients were generated using non-integrating episomal plasmids. In addition, to determine the functional significance of BA-susceptibility genes identified by genome-wide association studies (GWAS) in biliary development, a genome-editing approach was used to create iPSCs with defined mutations in these GWAS BA loci. Using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, isogenic iPSCs deficient in BA-associated genes (GPC1 and ADD3) were created from healthy iPSCs. Both the BA patient-iPSCs and the knock out (KO) iPSCs were studied for their in vitro biliary differentiation potential. These BA-specific iPSCs demonstrated significantly decreased formation of ductal structures, decreased expression of biliary markers including CK7, EpCAM, SOX9, CK19, AE2, and CFTR and increased fibrosis markers such as alpha smooth muscle actin, Loxl2, and Collagen1 compared to controls. Both the patient- and the KO-iPSCs also showed increased yes-associated protein (YAP, a marker of bile duct proliferation/fibrosis). Collagen and YAP were reduced by treatment with the anti-fibrogenic drug pentoxifylline. In summary, these BA-specific human iPSCs showed deficiency in biliary differentiation along with increased fibrosis, the 2 key disease features of BA. These iPSCs can provide new human BA models for understanding the molecular basis of abnormal biliary development and opportunities to identify drugs that have therapeutic effects on BA.

A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease.

Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with ß-thalassemia (ß-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of ß-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two ß-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in ß-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.

Derivation of a disease-specific human induced pluripotent stem cell line from a biliary atresia patient.

Biliary atresia (BA) is a common cause of pediatric end-stage liver disease. While its etiology is not yet clear, evidence has suggested that BA results from interactions between genetic susceptibility and environmental factors. Disease relevant human cellular models of BA will facilitate identification of both genetic and environmental factors that are important for disease prevention and treatment. Here we report the generation of a human induced pluripotent stem cell line from a BA patient using episomal vectors. Patient-specific BA iPSC lines provide valuable tools for disease mechanism study and drug development.

Generation of human iPSCs from an essential thrombocythemia patient carrying a V501L mutation in the MPL gene.

Activating point mutations in the MPL gene encoding the thrombopoietin receptor are found in 3%-10% of essential thrombocythemia (ET) and myelofibrosis patients. Here, we report the derivation of induced pluripotent stem cells (iPSCs) from an ET patient with a heterozygous MPL V501L mutation. Peripheral blood CD34+ progenitor cells were reprogrammed by transient plasmid expression of OCT4, SOX2, KLF4, c-MYC plus BCL2L1 (BCL-xL) genes. The derived line M494 carries a MPL V501L mutation, displays typical iPSC morphology and characteristics, are pluripotent and karyotypically normal. Upon differentiation, the iPSCs are able to differentiate into cells derived from three germ layers.

A hypomorphic PIGA gene mutation causes severe defects in neuron development and susceptibility to complement-mediated toxicity in a human iPSC model.

Mutations in genes involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis underlie a group of congenital syndromes characterized by severe neurodevelopmental defects. GPI anchored proteins have diverse roles in cell adhesion, signaling, metabolism and complement regulation. Over 30 enzymes are required for GPI anchor biosynthesis and PIGA is involved in the first step of this process. A hypomorphic mutation in the X-linked PIGA gene (c.1234C>T) causes multiple congenital anomalies hypotonia seizure syndrome 2 (MCAHS2), indicating that even partial reduction of GPI anchored proteins dramatically impairs central nervous system development, but the mechanism is unclear. Here, we established a human induced pluripotent stem cell (hiPSC) model containing the PIGAc.1234C>T mutation to study the effects of a hypomorphic allele of PIGA on neuronal development. Neuronal differentiation from neural progenitor cells generated by EB formation in PIGAc.1234C>T is significantly impaired with decreased proliferation, aberrant synapse formation and abnormal membrane depolarization. The results provide direct evidence for a critical role of GPI anchor proteins in early neurodevelopment. Furthermore, neural progenitors derived from PIGAc.1234C>T hiPSCs demonstrate increased susceptibility to complement-mediated cytotoxicity, suggesting that defective complement regulation may contribute to neurodevelopmental disorders.

Gene correction in patient-specific iPSCs for therapy development and disease modeling.

The discovery that mature cells can be reprogrammed to become pluripotent and the development of engineered endonucleases for enhancing genome editing are two of the most exciting and impactful technology advances in modern medicine and science. Human pluripotent stem cells have the potential to establish new model systems for studying human developmental biology and disease mechanisms. Gene correction in patient-specific iPSCs can also provide a novel source for autologous cell therapy. Although historically challenging, precise genome editing in human iPSCs is becoming more feasible with the development of new genome-editing tools, including ZFNs, TALENs, and CRISPR. iPSCs derived from patients of a variety of diseases have been edited to correct disease-associated mutations and to generate isogenic cell lines. After directed differentiation, many of the corrected iPSCs showed restored functionality and demonstrated their potential in cell replacement therapy. Genome-wide analyses of gene-corrected iPSCs have collectively demonstrated a high fidelity of the engineered endonucleases. Remaining challenges in clinical translation of these technologies include maintaining genome integrity of the iPSC clones and the differentiated cells. Given the rapid advances in genome-editing technologies, gene correction is no longer the bottleneck in developing iPSC-based gene and cell therapies; generating functional and transplantable cell types from iPSCs remains the biggest challenge needing to be addressed by the research field.

Efficient and Controlled Generation of 2D and 3D Bile Duct Tissue from Human Pluripotent Stem Cell-Derived Spheroids.

While in vitro liver tissue engineering has been increasingly studied during the last several years, presently engineered liver tissues lack the bile duct system. The lack of bile drainage not only hinders essential digestive functions of the liver, but also leads to accumulation of bile that is toxic to hepatocytes and known to cause liver cirrhosis. Clearly, generation of bile duct tissue is essential for engineering functional and healthy liver. Differentiation of human induced pluripotent stem cells (iPSCs) to bile duct tissue requires long and/or complex culture conditions, and has been inefficient so far. Towards generating a fully functional liver containing biliary system, we have developed defined and controlled conditions for efficient 2D and 3D bile duct epithelial tissue generation. A marker for multipotent liver progenitor in both adult human liver and ductal plate in human fetal liver, EpCAM, is highly expressed in hepatic spheroids generated from human iPSCs. The EpCAM high hepatic spheroids can, not only efficiently generate a monolayer of biliary epithelial cells (cholangiocytes), in a 2D differentiation condition, but also form functional ductal structures in a 3D condition. Importantly, this EpCAM high spheroid based biliary tissue generation is significantly faster than other existing methods and does not require cell sorting. In addition, we show that a knock-in CK7 reporter human iPSC line generated by CRISPR/Cas9 genome editing technology greatly facilitates the analysis of biliary differentiation. This new ductal differentiation method will provide a more efficient method of obtaining bile duct cells and tissues, which may facilitate engineering of complete and functional liver tissue in the future.

A Method for Genome Editing in Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSCs) hold great potential for regenerative medicine and currently are being used as a research tool for basic discovery and disease modeling. To evaluate the role of a single genetic variant, a system of genome editing is needed to precisely mutate any desired DNA sequence in isolation and measure its effect on phenotype when compared to the isogenic parental PSC from which it was derived. This protocol describes the general targeting schemes used by researchers to edit PSCs to knock out, knock-in, or precisely alter a single nucleotide, covering conditions for electroporation, clonal isolation, and screening of edited PSCs for the targeted mutation. These recent advances simplify the procedure for genome editing, allowing individual researchers to induce nearly any desired mutation to further study its function or to reverse a disease-causing variant for future applications in regenerative medicine.

Genome Editing in Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities.

Genome editing systems in novel therapies.

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

Modified Ham test for atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.