Nonstochastic reprogramming from a privileged somatic cell state.

Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:

Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

The Role of T Cells in Alzheimer's Disease Pathogenesis.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with memory decline and cognitive impairment, which is related to hallmark protein aggregates, amyloid-ß (Аß) plaques and neurofibrillary tangles; the latter are accumulated with hyperphosphorylated Tau protein. Immune cells play an important role in AD pathogenesis. Although the role of T cells in AD remains controversial, studies have shown that T cell deficiency is associated with increased AD pathology. In contrast, transplantation of T cells reduces AD pathology. T cells can help B cells generate anti-Ðß antibody to neutralize the toxin of Ðß and hyperphosphorylated Tau. T cells also activate macrophages to phagocytose misfolded proteins including Ðß and Tau. Recent data have also shown that AD animals have a damaged thymic microenvironment, especially thymic epithelial cells (TECs), resulting in decreased T cell numbers, which contribute to AD pathology. Therefore, regulation of T cell regeneration, for example by rejuvenating the thymic microenvironment, has the potential to be used in the treatment of AD.

An age classification model based on DNA methylation biomarkers of aging in human peripheral blood using random forest and artificial neural network.

Recent epigenetic studies have revealed a strong association between DNA methylation and aging and lifespan, which changes (increases or decreases) with age. Based on these, the construction of age prediction models associated with DNA methylation levels can be used to infer biological ages closer to the functional state of the organism. We downloaded methylation data from the Gene Expression Omnibus (GEO) public database for normal peripheral blood samples from people of different ages. We grouped the samples according to age (18-35 years and >50 years), screened the methylation sites that differed between the two groups, identified 44 differentially methylated sites, and subsequently obtained 11 age-related characteristic methylation sites using the random forest method. Then, we constructed an age classification model with these 11 characteristic methylation sites using an artificial neural network and evaluated its efficacy. The age classification model was constructed by an artificial neural network and its efficacy was evaluated. The model predicted an area under the curve (AUC) of 0.97 in the validation set and accurately distinguished between those aged 18-35 and >50 years. Furthermore, the levels of these 11 characteristic methylation sites also differed significantly between the two sets of samples in the validation set, including six newly identified age-related methylation sites (P<0.001). Finally, we constructed a multifactor regulatory network based on the corresponding genes of age-related methylation sites to reveal the transcriptional and post-transcriptional regulation patterns. As a result of the increasing problem of aging, the age classification model we constructed allows us to accurately distinguish different age groups at the molecular level, which will be more predictive than chronological age for assessing individual aging and future health status.

Regulatory role of Chitinase 3-like 1 gene in papillary thyroid carcinoma proved by integration analyses of single-cell sequencing with cohort and experimental validations.

Papillary thyroid carcinoma (PTC) is one of the most common thyroid carcinomas. The gross extrathyroidal extension and extensive metastases of PTC lead to high rates of recurrence and poor clinical outcomes. However, the mechanisms underlying PTC development are poorly understood. In this study, using single-cell RNA sequencing, the transcriptome profiles of two PTC patients were addressed, including PTC1 with low malignancy and good prognosis and PTC2 with high malignancy and poor prognosis. We found that epithelial subcluster Epi02 was the most associated with the malignant development of PTC cells, with which the fold change of Chitinase 3-like 1 (CHI3L1) is on the top of the differentially expressed genes between PTC1 and PTC2 (P < 0.001). However CHI3L1 is rarely investigated in PTC as far. We then studied its role in PTC with a series of experiments. Firstly, qRT-PCR analysis of 14 PTC patients showed that the expression of CHI3L1 was positively correlated with malignancy. In addition, overexpression or silencing of CHI3L1 in TPC-1 cells, a PTC cell line, cultured in vitro showed that the proliferation, invasion, and metastasis of the cells were promoted or alleviated by CHI3L1. Further, immunohistochemistry analysis of 110 PTC cases revealed a significant relationship between CHI3L1 protein expression and PTC progression, especially the T (P < 0.001), N (P < 0.001), M stages (P = 0.007) and gross ETE (P < 0.001). Together, our results prove that CHI3L1 is a positive regulator of malignant development of PTC, and it promotes proliferation, invasion, and metastasis of PTC cells. Our study improves understanding of the molecular mechanisms underlying the progression of PTC and provides new insights for the clinical diagnosis and treatment of PTC.

Sample-multiplexing approaches for single-cell sequencing.

Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing, high experimental costs, ambiguous identification of true single cells, and technical batch effects. Here, we introduce sample-multiplexing approaches for single-cell sequencing in transcriptomics, epigenomics, genomics, and multiomics. In single-cell transcriptomics, sample multiplexing uses variants of native or artificial features as sample markers, enabling sample pooling and decoding. Such features include: (1) natural genetic variation, (2) nucleotide-barcode anchoring on cellular or nuclear membranes, (3) nucleotide-barcode internalization to the cytoplasm or nucleus, (4) vector-based barcode expression in cells, and (5) nucleotide-barcode incorporation during library construction. Other single-cell omics methods are based on similar concepts, particularly single-cell combinatorial indexing. These methods overcome current challenges, while enabling super-loading of single cells. Finally, selection guidelines are presented that can accelerate technological application.

Putative regulators for the continuum of erythroid differentiation revealed by single-cell transcriptome of human BM and UCB cells.

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34-CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP, and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation.

Single-cell transcriptomic sequencing analyses of cell heterogeneity during osteogenesis of human adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are known for their multilineage differentiation potential with immune-modulatory properties. The molecular underpinnings of differentiation remain largely undefined. In this study, we investigated the cellular and molecular features of chemically induced osteogenesis from MSC isolated from human adipose tissue (human adipose MSCs, hAMSCs) using single-cell RNA-sequencing (scRNA-seq). We found that a near complete differentiation of osteogenic clusters from hAMSCs under a directional induction. Both groups of cells are heterogeneous, and some of the hAMSCs cells are intrinsically prepared for osteogenesis, while variant OS clusters seems in cooperation with a due division of the general function. We identified a set of genes related to cell stress response highly expressed during the differentiation. We also characterized a series of transitional transcriptional waves throughout the process from hAMSCs to osteoblast and specified the unique gene networks and epigenetic status as key markers of osteogenesis.

Lack of tumorigenesis and protumorigenic activity of human umbilical cord mesenchymal stem cells in NOD SCID mice.

BACKGROUND: The tumorigenesis of infused umbilical cord mesenchymal stem cells (UC-MSCs) is being preclinically evaluated. METHODS: We observed tumor formation in NOD SCID mice after a single subcutaneous injection of hUC-MSCs and the effect of these cells on tumor growth in tumor-bearing mice. Three generations (P5, P7, and P10) of hUC-MSCs (1 × 107) from two donors (hUC-MSC1 and hUC-MSC2) were inoculated subcutaneously into NOD SCID mice. Subcutaneous transplantation models were established in NOD SCID mice with human cervical cancer HeLa cells (solid tumor) and human B cell lymphoma Raji cells (hematological tumor). Then, the animals were euthanized, gross dissection was performed, and tissues were collected. Various organs were observed microscopically to identify pathological changes and tumor metastasis. RESULTS: In the tumorigenesis experiment, no general anatomical abnormalities were observed. In the tumor promotion experiment, some animals in the HeLa groups experienced tumor rupture, and one animal died in each of the low- and medium-dose hUC-MSC groups. The results may have occurred due to the longer feeding time, and the tumor may have caused spontaneous infection and death. Pathological examination revealed no metastasis to distant organs in any group. In the Raji tumor model, some animals in each group experienced tumor rupture, and one animal in the medium-dose hUC-MSC group died, perhaps due to increased tumor malignancy. Thus, hUC-MSCs neither promoted nor inhibited tumor growth. No cancer cell metastasis was observed in the heart, liver, spleen, lungs, kidneys or other important organs, except that pulmonary venule metastasis was observed in 1 animal in the model group. CONCLUSIONS: Injected hUC-MSCs were not tumorigenic and did not significantly promote or inhibit solid or hematological tumor growth or metastasis in NOD SCID mice.

Establishment of a Systemic Inflammatory Response Syndrome Model and Evaluation of the Efficacy of Umbilical Cord Mesenchymal Stem Cell Transplantation.

Based on the characteristics of modern weapon injury, a repetitive model of traumatic systemic inflammatory response syndrome (SIRS) and an evaluation system were established. The models were treated with GFP-labeled tree shrew umbilical cord mesenchymal stem cells (UCMSCs). Forty out of 50 tree shrews were used to make a unilateral femoral comminuted fracture. Lipopolysaccharide was injected intravenously to create a traumatic SIRS model. The other 10 shrews were used as normal controls. After the model was established for 10 days, 20 tree shrews were injected intravenously with GFP-labeled UCMSCs, and 18 tree shrews were not injected as the model control group. The distribution of GFP-labeled cells in vivo was measured at 2 and 10 days after injection. Twenty days after treatment, the model group, the normal control group, and the treatment group were taken to observe the pathological changes in each tissue, and blood samples were taken for the changes in liver, renal, and heart function. Distribution of GFP-positive cells was observed in all tissues at 2 and 10 days after injection. After treatment, the HE staining results of the treatment group were close to those of the normal group, and the model group had a certain degree of lesions. The results of liver, renal, and heart function tests in the treatment group were returned to normal, and the results in the model group were abnormally increased. UCMSCs have a certain effect on the treatment of traumatic SIRS and provide a new technical solution for modern weapon trauma treatment.

MustSeq, an alternative approach for multiplexible strand-specific 3' end sequencing of mRNA transcriptome confers high efficiency and practicality.

RNA-seq has been widely used to reveal the molecular mechanism of variants of life process. We have developed an alternative method, MustSeq, which generates multiple second strands along a single 1st strand cDNA by random-priming initiation, immediately after reverse transcription for each RNA extract using sample-barcoded poly-dT primers, then 3' ends-enriching PCR is applied to construct the library. Unlike the conventional RNA seq, MustSeq avoids procedures such as mRNA isolation, fragmentation and RNA 5'-end capture, enables early pooling of multiple samples, and requires only one twentieth of sequencing reads of full-length sequencing. We demonstrate the power and features of MustSeq comparing with TruSeq and NEBNext RNA-seq, two conventional full-length methods and QuantSeq, an industrial 3' end method. In cancer cell lines, the reads distribution of CDS-exon as well as genes, lncRNAs and GO terms detected by MustSeq are closer than QuantSeq to TruSeq. In mouse hepatocarcinoma and healthy livers, MustSeq enriches the same pathways as by NEBNext, and reveals the molecular profile of carcinogenesis. Overall MustSeq is a robust and accurate RNA-seq method allowing efficient library construction, sequencing and analysis, particularly valuable for analysis of differentially expressed genes with a large number of samples. MustSeq will greatly accelerate the application of bulk RNA-seq on different fields, and potentially applicable for single cell RNA-seq.

Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation.

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-ß signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

Identification of a distinct luminal subgroup diagnosing and stratifying early stage prostate cancer by tissue-based single-cell RNA sequencing.

BACKGROUND: The highly intra-tumoral heterogeneity and complex cell origination of prostate cancer greatly limits the utility of traditional bulk RNA sequencing in finding better biomarker for disease diagnosis and stratification. Tissue specimens based single-cell RNA sequencing holds great promise for identification of novel biomarkers. However, this technique has yet been used in the study of prostate cancer heterogeneity. METHODS: Cell types and the corresponding marker genes were identified by single-cell RNA sequencing. Malignant states of different clusters were evaluated by copy number variation analysis and differentially expressed genes of pseudo-bulks sequencing. Diagnosis and stratification of prostate cancer was estimated by receiver operating characteristic curves of marker genes. Expression characteristics of marker genes were verified by immunostaining. RESULTS: Fifteen cell groups including three luminal clusters with different expression profiles were identified in prostate cancer tissues. The luminal cluster with the highest copy number variation level and marker genes enriched in prostate cancer-related metabolic processes was considered the malignant cluster. This cluster contained a distinct subgroup with high expression level of prostate cancer biomarkers and a strong distinguishing ability of normal and cancerous prostates across different pathology grading. In addition, we identified another marker gene, Hepsin (HPN), with a 0.930 area under the curve score distinguishing normal tissue from prostate cancer lesion. This finding was further validated by immunostaining of HPN in prostate cancer tissue array. CONCLUSION: Our findings provide a valuable resource for interpreting tumor heterogeneity in prostate cancer, and a novel candidate marker for prostate cancer management.

The Genetics Analysis of Molecular Pathogenesis for Alzheimer's Disease.

INTRODUCTION: The molecular pathogenesis of Alzheimer's disease (AD) is still not clear, and the relationship between gene expression profile for different brain regions has not been studied. OBJECTIVE: Bioinformatic analysis at the genetic level has become the best way for the pathogenesis research of AD, which can analyze the abovementioned relationship. METHODS: In this study, the datasets of AD were obtained from the Gene Expression Omnibus (GEO), and Qlucore Omics Explorer (QOE) software was used to screen differentially expressed genes of GSE36980 and GSE9770 and verify gene expression of GSE63060. The Gene Ontology (GO) function enrichment analysis of these selected genes was conducted by Database for Annotation, Visualization, and Integrated Discovery (DAVID), and then the gene/protein interaction network was established by STRING to find the related proteins. R language was used for drafting maps and plots. RESULTS: There were 20 differentially expressed genes related to AD selected from GSE36980 (p = 6.2e-6, q = 2.9422e-4) and GSE9770 (p = 3.3e-4, q = 0.016606). Their expression levels of the AD group were lower than those in the control group and varied among different brain regions. Cellular morphogenesis and establishment or maintenance of cell polarity were enriched, and LRRTM1 and RASAL1 were identified by the integration network. Moreover, the analysis of GSE63060 verified the expression level of LRRTM1 and RASAL1 in Alzheimer's patients, which was much lower than that in normal people aged >65 years. CONCLUSIONS: The pathogenesis of AD at molecular levels may link to cell membrane structures and signal transduction; hence, a list of 20 genes, including LRRTM1 and RASAL1,potentially are important for the discovery of treatment target or molecular marker of AD.

Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors.

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.

The role of HMGB1 in BMSC transplantation for treating MODS in rats.

The effect of bone marrow mesenchymal stem cells (BMSCs) in treatment for multiple organ dysfunction syndrome (MODS) remains unknown and the mechanism is still unclear. Therefore, the goal of this study is to investigate the effects of intracellular high mobility group box 1 protein (HMGB1) on BMSCs treating for MODS. The rats were given 15% blood loss plus 1 mg/kg lipopolysaccharide (LPS) via lower extremity superficial venous, then randomly allocated into four groups: sham group, MODS group, MODS plus BMSC group, MODS plus ethyl pyruvate (EP) group, MODS plus BMSCs plus EP group. Twenty-four hours later, rats in groups were sacrificed and then the blood and tissues were collected to evaluate the changes of tissue histopathology, cell apoptosis, inflammation level and organ function. The HGMB1 expression was monitored by RT-qPCR and Western blot. The expression of RAGE/TLR2/TLR4 and NF-κB at the protein levels was also assessed. BMSCs and/or EP exhibits an outstanding protective effect against LPS-induced histopathological injury by improving cell apoptosis, inflammatory response and the organ dysfunction but no effect on BMSC homing to the injury site. Moreover, BMSCs and/or EP inhibited LPS-induced upregulation of HMGB1, RAGE, TLR2 and TLR4 expression at protein levels and compromised p65 phosphorylation in the rat model of MODS. These findings suggest that HMGB1 is involved in BMSC treatment for MODS, through regulation of the TLR2, TLR4-mediated NF-κB signal pathway. It suggests that HMGB1 is an attractive potential target for the development of new therapeutic strategies for MODS.

Telomere shortening activates TGF-ß/Smads signaling in lungs and enhances both lipopolysaccharide and bleomycin-induced pulmonary fibrosis.

Telomere shortening is associated with idiopathic pulmonary fibrosis (IPF), a high-morbidity and high-mortality lung disease of unknown etiology. However, the underlying mechanisms remain largely unclear. In this study, wild-type (WT) mice with normal telomeres and generation 3 (G3) or G2 telomerase RNA component (TERC) knockout Terc-/- mice with short telomeres were treated with and without lipopolysaccharide (LPS) or bleomycin by intratracheal injection. We show that under LPS induction, G3 Terc-/- mice develop aggravated pulmonary fibrosis as indicated by significantly increased α-SMA, collagen I and hydroxyproline content. Interestingly, TGF-ß/Smads signaling is markedly activated in the lungs of G3 Terc-/- mice, as indicated by markedly elevated levels of phosphorylated Smad3 and TGF-ß1, compared with those of WT mice. This TGF-ß/Smads signaling activation is significantly increased in the lungs of LPS-treated G3 Terc-/- mice compared with those of LPS-treated WT or untreated G3 Terc-/- mice. A similar pattern of TGF-ß/Smads signaling activation and the enhancing role of telomere shortening in pulmonary fibrosis are also confirmed in bleomycin-induced model. Moreover, LPS challenge produced more present cellular senescence, apoptosis and infiltration of innate immune cells, including macrophages and neutrophils in the lungs of G3 Terc-/- mice, compared with WT mice. To our knowledge, this is the first time to report telomere shortening activated TGF-ß/Smads signaling in lungs. Our data suggest that telomere shortening cooperated with environment-induced lung injury accelerates the development of pulmonary fibrosis, and telomere shortening confers an inherent enhancing factor to the genesis of IPF through activation of TGF-ß/Smads signaling.

Next-generation molecular diagnosis: single-cell sequencing from bench to bedside.

Single-cell sequencing (SCS) is a fast-growing, exciting field in genomic medicine. It enables the high-resolution study of cellular heterogeneity, and reveals the molecular basis of complicated systems, which facilitates the identification of new biomarkers for diagnosis and for targeting therapies. It also directly promotes the next generation of genomic medicine because of its ultra-high resolution and sensitivity that allows for the non-invasive and early detection of abnormalities, such as aneuploidy, chromosomal translocation, and single-gene disorders. This review provides an overview of the current progress and prospects for the diagnostic applications of SCS, specifically in pre-implantation genetic diagnosis/screening, non-invasive prenatal diagnosis, and analysis of circulating tumor cells. These analyses will accelerate the early and precise control of germline- or somatic-mutation-based diseases, particularly single-gene disorders, chromosome abnormalities, and cancers.

Chronic Toxicity Test in Cynomolgus Monkeys For 98 Days with Repeated Intravenous Infusion of Cynomolgus Umbilical Cord Mesenchymal Stem Cells.

BACKGROUND/AIMS: Stem cell-based therapy is attractive in many clinical studies, but current data on the safety of stem cell applications remains inadequate. This study observed the safety, immunological effect of cynomolgus monkey umbilical cord mesenchymal stem cells (mUC-MSCs) injected into cynomolgus monkeys, in order to evaluate the safety of human umbilical cord mesenchymal stem cells (hUC-MSCs) prepared for human clinical application. METHODS: Eighteen cynomolgus monkeys were divided into three groups. Group 1 is control group, Group 2 is low-dose group, Group 3 is high-dose group. After repeated administrations of mUC-MSCs, cynomolgus monkeys were observed for possible toxic reactions. RESULTS: During the experiment, no animal died. There were no toxicological abnormalities in body weight, body temperature, electrocardiogram, coagulation and pathology. In the groups 2 and 3, AST and CK transiently increased, and serum inorganic P slightly decreased. All animals were able to recover at 28 days after the infusion was stopped. In the groups 2 and 3, CD3+ and IL-6 levels significantly increased, and recovery was after 28 days of infusion. There were no obvious pathological changes associated with the infusion of cells in the general and microscopic examinations. CONCLUSIONS: The safe dosage of repeated intravenous infusion of mUC-MSCs in cynomolgus monkeys is 1.0 × 107/kg, which is 10 times of that in clinical human use.