Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. METHODS: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. RESULTS: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. CONCLUSIONS: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Long Non-Coding RNA (lncRNA) CRNDE Regulated Lipopolysaccharides (LPS)-Induced MRC-5 Inflammation Injury Through Targeting MiR-141.

BACKGROUND Pneumonia is a common disease with high morbidity and even death. In our country, pneumonia is the leading cause of child death. Therefore, research on the pathogenesis of pneumonia can help improve the treatment of pneumonia. Long non-coding RNA (lncRNA) is an important regulator of disease development, and its regulatory mechanism is closely related to cellular processes. However, the function and regulatory network of lncRNA is not fully elucidated in pneumonia. MATERIAL AND METHODS Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of CRNDE and miR-141 in lipopolysaccharides (LPS)-induced MRC-5 cells and pneumonia tissues. MTT (3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyl-2-tetrazolium) assay was used to assess cell proliferation. Flow cytometry assay was performed to detect cell apoptosis in LPS-induced MRC-5 cells. Enzyme-linked immunosorbent assay and western blot were used to measure the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-alpha, respectively. In addition, luciferase reporter assay and RNA immunoprecipitation (RIP) assay were applied to prove the relationship between CRNDE and miR-141. RESULTS In this study, we found that CRNDE expression was induced in LPS-induced MRC-5 cells and pneumonia tissues. Moreover, miR-141 expression was low in LPS-induced MRC-5 cells and was verified was a target miRNA of CRNDE by using luciferase reporter assay and RIP assay. The downregulation of CRNDE and upregulation of miR-141 promoted cell viability, inhibited cell apoptosis, as well as decreased the levels of IL-1ß, IL-6, and TNF-alpha. Moreover, we demonstrated that si-CRNDE transfection increased cell viability and suppressed cell apoptosis and the levels of IL-1ß, IL-6, and TNF-alpha, which were alleviated by anti-miR-141 transfection in LPS-induced MRC-5 cells. CONCLUSIONS In this study, we found that downregulation of CRNDE and upregulation of miR-141 inhibited cell apoptosis and inflammation response and promoted cell viability in LPS-induced MRC-5 cells. Low CRNDE expression increased cell growth and suppressed inflammation response, which was impaired by inhibition of miR-141. These results suggested that a novel therapeutic target was found in pneumonia treatment.

Critical role of Lin28-TNFR2 signalling in cardiac stem cell activation and differentiation.

Tumour necrotic factor receptor-2 (TNFR2) has been to be cardiac-protective and is expressed in cardiac progenitor cells. Our goal is to define the mechanism for TNFR2-mediated cardiac stem cell activation and differentiation. By employing a protocol of in vitro cardiac stem cell (CSC) differentiation from human inducible pluripotent stem cell (hiPSC), we show that expression of TNFR2 precedes expression of CSC markers followed by expression of mature cardiomyocyte proteins. Activation of TNFR2 by a specific agonist promotes whereas inhibition of TNFR2 by neutralizing antibody diminishes hiPSC-based CSC differentiation. Interestingly, pluripotent cell factor RNA-binding protein Lin28 enhances TNFR2 protein expression in early CSC activation by directly binding to a conserved Lin28-motif within the 3'UTR of Tnfr2 mRNA. Furthermore, inhibition of Lin28 blunts TNFR2 expression and TNFR2-dependent CSC activation and differentiation. Our study demonstrates a critical role of Lin28-TNFR2 axis in CSC activation and survival, providing a novel strategy to enhance stem cell-based therapy for the ischaemic heart diseases.

Characterization of the mammalian miRNA turnover landscape.

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.

Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing.

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation-N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)-were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.

Relationship between HPV-16 infection and the progression of vaginal intraepithelial neoplasia.

OBJECTIVE: To explore the relationship between infection with human papillomavirus (HPV)-16 and the development of vaginal intraepithelial neoplasia (VaIN). METHODS: This is a retrospective study. 78 patients with suspected VaIN admitted to the gynecologic clinic of Affiliated Hospital of Guilin Medical College from August 2016 to December 2020 who were confirmed to have HPV-16 infection by HPV rapid flow-through hybridization method were selected as the research subjects. The copy numbers of HPV-16 early genes E2 and E6 were detected by quantitative real-time PCR amplification to analyze the integration status of the virus. RESULTS: The episomal form of HPV-16 exists in all levels of VaIN. As the pathological level of VaIN increased, the episomal form of HPV-16 gradually decreased, and a disruption of the E2 gene became more frequent. However, there was no significant difference between different levels of VaIN (P>0.05). With the increased severity of cytology results, the percentage of the episomal form of HPV-16 decreased from 76.47% in atypical squamous cells of undetermined significance (ASC-US) to 44.44% in the high-grade squamous intraepithelial lesion (HSIL) (χ2 =4.780, P<0.05). However, the integrated form of HPV-16 did not increase significantly as the severity of cytology increased (χ2=2.215, P>0.05). CONCLUSION: HPV gene integration may occur before the onset of VaIN. However, the occurrence of HPV-16 integration is not a risk factor leading to the malignant progression of VaIN. An E2 gene disruption is more common in the early events after HPV-16 infection. HPV-16 gene integration may be the main reason for persistent HPV-16 infection.

Comparative efficacy of pulmonary surfactant in respiratory distress syndrome in preterm infants: a Bayesian network meta-analysis.

Introduction: The comparative efficacy of pulmonary surfactant in the treatment of respiratory distress syndrome in preterm infants remains unclear. We aimed to evaluate the effectiveness of different pulmonary surfactant in the treatment of respiratory distress syndrome in preterm infants and to provide an evidence-based reference for clinical use. Material and methods: MEDLINE, Embase, The Cochrane Library, and Clinical Trials databases were electronically searched from inception to January 2019. Two reviewers independently screened literature and extracted data, and then R and RevMan 5.3 software packages were used to perform network meta-analysis. Results: The relative risk of respiratory distress syndrome in preterm infants associated with six different pulmonary surfactant was analysed, including beractant (Survanta), surfactant A (Alveofact), calfactant (Infasurf), poractant (Curosurf), lucinactant (Surfaxin), and colfosceril (Exosurf). Patients with the following drugs appeared to have significantly reduced mortality of respiratory distress syndrome compare with beractant: surfactant A (OR = 0.53, 95% CI: 0.31-0.90), calfactant (OR = 0.91, 95% CI: 0.85-0.97), poractant (OR = 0.72, 95% CI: 0.67-0.77), lucinactant (OR = 0.80, 95% CI: 0.71-0.90), and colfosceril (OR = 0.93, 95% CI: 0.87-0.99). The SUCRA (surface under the cumulative ranking) values for each of the drugs were: beractant (8.9%), surfactant A (93.8%), calfactant (40.3%), poractant (65.4%), lucinactant (59.8%), and colfosceril (31.6%). Conclusions: Compared with beractant, other pulmonary surfactants are more effective to reduce the mortality of respiratory distress syndrome in preterm infants. Surfactant A drugs appeared to have the best efficacy in reducing mortality of respiratory distress syndrome in preterm infants.

High-efficiency siRNA-based gene knockdown in human embryonic stem cells.

Loss-of-function studies in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) via nonviral approaches have been largely unsuccessful. Here we report a simple and cost-effective method for high-efficiency delivery of plasmids and siRNAs into hESCs and iPSCs. Using this method for siRNA delivery, we achieve >90% reduction in the expression of the stem cell factors Oct4 and Lin28, and observe cell morphological and staining pattern changes, characteristics of hESC differentiation, as a result of Oct4 knockdown.

Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells.

Lin28 acts as a repressor of microRNA processing and as a post-transcriptional regulatory factor for a subset of mRNAs. Here we report that in human embryonic stem cells Lin28 facilitates the expression of the pivotal pluripotency factor Oct4 at the post-transcriptional level. We provide evidence that Lin28 binds Oct4 mRNA directly through high affinity sites within its coding region and that an interaction between Lin28 and RNA helicase A (RHA) may play a part in the observed regulation. We further demonstrate that decreasing RHA levels impairs Lin28-dependent stimulation of translation in a reporter system. Taken together with previous studies showing that RHA is required for efficient translation of a specific class of mRNAs, these findings suggest a novel mechanism by which Lin28 may affect target mRNA expression and represent the first evidence of post-transcriptional regulation of Oct4 expression by Lin28 in human embryonic stem cells.

The role of FYCO1-dependent autophagy in lens fiber cell differentiation.

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1-/-) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1-/- mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1-/- mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis.Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type.

Role for MKL1 in megakaryocytic maturation.

Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34(+) cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

The Effect of lncRNA SNHG3 Overexpression on Lung Adenocarcinoma by Regulating the Expression of miR-890.

The lncRNA small nucleolar host gene 3 (SNHG3) was discovered to play an important role in the occurrence and development of lung adenocarcinoma (LUAD). However, the underlying molecular mechanism of SNHG3 in LUAD remains unclear. In the present study, SNHG3 expression levels in LUAD tissues and cell lines were analyzed using reverse transcription-quantitative PCR. The effects of SNHG3 on the proliferation, apoptosis, migration, and invasion of LUAD cells were determined using Cell Counting Kit-8, colony formation, flow cytometry, wound healing, and Transwell chamber assays, respectively. The specific underlying mechanism of SNHG3 in LUAD was investigated using bioinformatics analysis and a dual luciferase reporter assay. The results revealed that SNHG3 expression levels were downregulated in LUAD tissues and cell lines. Functionally, SNHG3 overexpression suppressed the proliferation, migration, and invasion of LUAD cells, while promoting apoptosis. Mechanistically, microRNA- (miR-) 890 was identified as a potential target of SNHG3, and its expression was negatively regulated by SNHG3. Notably, SNHG3 was found to promote LUAD progression by targeting miR-890. In conclusion, the findings of the present study revealed that lncRNA SNHG3 promoted the occurrence and progression of LUAD by regulating miR-890 expression.

Comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies.

The ocular lens serves as an excellent system to investigate the intricate details of development and differentiation. Generation of lentoid bodies or lens-like structures using pluripotent stem cells is important for understanding the processes critical for lens morphogenesis and the mechanism of cataractogenesis. We previously reported the generation of peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs). Here, we report generation of lentoid bodies from human embryonic stem cells (hESCs) and (PBMC)-originated, iPSCs employing the "fried egg" method with brief modifications. The ultrastructure analysis of hESC- and iPSC-derived lentoid bodies identified closely packed lens epithelial- and differentiating fiber-like cells. In addition, we performed RNA sequencing (RNA-Seq) based transcriptome profiling of hESC- and iPSC-derived lentoid bodies at differentiation day 25. Next-generation RNA sequencing (RNA-Seq) of hESC- and iPSC-derived lentoid bodies detected expression (≥0.659 RPKM) of 13,975 and 14,003 genes, respectively. Comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies revealed 13,563 (>96%) genes common in both datasets. Among the genes common in both transcriptome datasets, 12,856 (~95%) exhibited a quantitatively similar expression profile. Next, we compared the mouse lens epithelial and fiber cell transcriptomes with hESC- and iPSC-derived lentoid bodies transcriptomes and identified > 96% overlap with lentoid body transcriptomes. In conclusion, we report first-time comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies at differentiation day 25.

Generation and Proteome Profiling of PBMC-Originated, iPSC-Derived Corneal Endothelial Cells.

Purpose: Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. Methods: We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry-based proteome sequencing. Results: The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry-based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. Conclusions: We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

OBJECTIVE: To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. MATERIALS AND METHODS: Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. RESULTS: CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. CONCLUSIONS: We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development.

OBJECTIVE: To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells. MATERIALS AND METHODS: FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction. RESULTS: Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined. CONCLUSIONS: FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells.

There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications.

Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family.

Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.

Small-diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells.

Despite the progress made thus far in the generation of small-diameter vascular grafts, cell sourcing still remains a problem. Human embryonic stem cells (hESCs) present an exciting new cell source for the regeneration applications due to their high proliferative and differentiation capabilities. In this study, the feasibility of creating small-diameter vascular constructs using smooth muscle cells (SMCs) differentiated from hESC-derived mesenchymal cells was evaluated. In vitro experiments confirmed the ability of these cells to differentiate into smooth muscle actin- and calponin-expressing SMCs in the presence of known inducers, such as transforming growth factor beta. Human vessel walls were constructed by culturing these cells in a bioreactor system under pulsatile conditions for 8 weeks. Histological analysis showed that vessel grafts had similarities to their native counterparts in terms of cellularity and SMC marker expression. However, markers of cartilage and bone tissue were also detected, thus raising questions about stable lineage commitment during differentiation and calling for more stringent analysis of differentiating cell populations.

Engineering a blood-retinal barrier with human embryonic stem cell-derived retinal pigment epithelium: transcriptome and functional analysis.

Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.