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1.  Generation of Haploid Spermatids with Fertilization and Development Capacity from Human Spermatogonial Stem Cells of Cryptorchid Patients 
Stem Cell Reports  2014;3(4):663-675.
Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.
Graphical Abstract
•Spermatogonia remain whereas meiotic male germ cells are rare in cryptorchid patients•Human SSCs of cryptorchid patients differentiate into phenotypic haploid spermatids•Round spermatids derived from human SSCs have fertilization and development capacity•Distinct gene profiles exist in embryos from round spermatid and somatic cell nuclei
He, Li, and colleagues showed that spermatogonia remained whereas meiotic germ cells were rare or lost in cryptorchid patients. SSCs of cryptorchid patients were induced to differentiate into cells with phenotypic, DNA content, and fertilization and development potentials of haploid spermatids. These data demonstrate the generation of functional and autologous male gametes for treating male infertility in azoospermia patients.
PMCID: PMC4223697  PMID: 25358793
2.  Nodal Promotes the Self-Renewal of Human Colon Cancer Stem Cells via an Autocrine Manner through Smad2/3 Signaling Pathway 
BioMed Research International  2014;2014:364134.
Colorectal cancer is one of the most common and fatal tumors. However, molecular mechanisms underlying carcinogenesis of colorectal cancer remain largely undefined. Here, we explored the expression and function of Nodal in colon cancer stem cells (CCSCs). Nodal and its receptors were present in numerous human colorectal cancer cell lines. NODAL and ALK-4 were coexpressed in human colon cancerous tissues, and NODAL, CD24, and CD44, markers for CCSCs, were expressed at higher levels in human colon cancerous tissues than adjacent noncancerous colon tissues. Human CCSCs were isolated by magnetic activated cell sorting using anti-CD24 and anti-CD44. Nodal transcript and protein were hardly detectable in CD44- or CD24-negative human colorectal cancer cell lines, whereas Nodal and its receptors were present in CCSCs. Notably, Nodal facilitated spheroid formation of human CCSCs, and phosphorylation of Smad2 and Smad3 was activated by Nodal in cells of spheres derived from human CCSCs. Collectively, these results suggest that Nodal promotes the self-renewal of human CCSCs and mediate carcinogenesis of human colorectal cancer via an autocrine manner through Smad2/3 pathway. This study provides a novel insight into molecular mechanisms controlling fate of human CCSCs and offers new targets for gene therapy of human colorectal cancer.
PMCID: PMC3947734  PMID: 24696849
3.  Direct transdifferentiation of spermatogonial stem cells to morphological, phenotypic and functional hepatocyte-like cells via the ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E 
Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state.
SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Tat, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs.
SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming.
PMCID: PMC3848919  PMID: 24047406
Spermatogonial stem cells; Direct transdifferentiation; Hepatic stem cells; Mature hepatocytes; Morphology and phenotype; Function; ERK1/2 and Smad2/3 signaling pathways
4.  Intronic Splicing Enhancers, Cognate Splicing Factors and Context Dependent Regulation Rules 
Nature structural & molecular biology  2012;19(10):1044-1052.
Most human genes produce multiple splicing isoforms with distinct functions. To systematically understand splicing regulation, we conducted an unbiased screen and identified >100 intronic splicing enhancers (ISEs) that were clustered by sequence similarity into six groups. All ISEs functioned in another cell type and heterologous introns, and their distribution and conservation patterns in different pre-mRNA regions are similar to exonic splicing silencers. Consistently all ISEs inhibited use of splice sites from exonic locations. The putative trans-factors of each ISE group were identified and validated. Five distinct ISE motifs were recognized by hnRNP H and F whose C-terminal domains were sufficient to render context-dependent activities of ISEs. The sixth group was controlled by factors that either activate or suppress splicing. This work provided a comprehensive picture of general ISE activities and provided new models of how a single element can function oppositely depending on its locations and binding factors.
PMCID: PMC3753194  PMID: 22983564
splicing regulation; splicing factors; RNA binding protein; context dependent activity
5.  A Complex Network of Factors with Overlapping Affinities Repress Splicing through Intronic Elements 
To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from intron. Each motif was validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs based on their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, recovering 24 known and novel splicing regulators. Specific domains in selected factors were sufficient to confer ISS activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, revealing a complex, overlapping network of protein:RNA interactions. This arrangement enables individual cis-element to function differently in distinct cellular contexts depending on the spectrum of regulatory factors present.
PMCID: PMC3537874  PMID: 23241926
splicing regulation; splicing factors; intronic splicing silencers; RNA binding protein; context dependent activity
6.  Differentiation of Induced Pluripotent Stem Cells into Male Germ Cells In Vitro through Embryoid Body Formation and Retinoic Acid or Testosterone Induction 
BioMed Research International  2012;2013:608728.
Generation of germ cells from pluripotent stem cells in vitro could have great application for treating infertility and provides an excellent model for uncovering molecular mechanisms controlling gametogenesis. In this study, we explored the differentiation potential of mouse induced pluripotent stem (iPS) cells towards male germ cells. Embryoid body formation and retinoic acid/testosterone induction were applied to promote differentiation of mouse iPS cells into male germ cells in vitro. Quantitative RT-PCR and immunoflourescence were performed to characterize the iPS cell differentiation process, and notably there were different temporal expression profiles of male germ cell-associated genes. The expression of proteins, including MVH, CDH1, and SCP3, was remarkably increased. mRNA expression of Stra8, Odf2, Act, and Prm1 was upregulated in iPS cells by retinoic acid or testosterone induction, whereas Oct-4 transcription was reduced in these cells compared to the controls. Hormones were also measured in the EB medium. DNA content analysis by flow cytometry revealed that iPS cells could differentiate into haploid cells through retinoic acid or testosterone treatment. Collectively, our results suggest that mouse iPS cells possess the potency to differentiate into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction.
PMCID: PMC3591174  PMID: 23509752
7.  Virus-induced gene-silencing in wheat spikes and grains and its application in functional analysis of HMW-GS-encoding genes 
BMC Plant Biology  2012;12:141.
The Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated. In this study, we explored the feasibility of using BSMV for gene silencing in wheat spikes or grains.
Apparent photobleaching on the spikes infected with BSMV:PDS at heading stage was observed after13 days post inoculation (dpi), and persisted until 30dpi, while the spikes inoculated with BSMV:00 remained green during the same period. Grains of BSMV:PDS infected spikes also exhibited photobleaching. Molecular analysis indicated that photobleached spikes or grains resulted from the reduction of endogenous PDS transcript abundances, suggesting that BSMV:PDS was able to induce PDS silencing in wheat spikes and grains. Inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing of PDS in wheat spikes. Furthermore, we used the BSMV-based system to reduce the transcript level of 1Bx14, a gene encoding for High-molecular-weight glutenin subunit 1Bx14 (HMW-GS 1Bx14), by 97 % in the grains of the BSMV:1Bx14 infected spikes at 15dpi, compared with that in BSMV:00 infected spikes, and the reduction persisted until at least 25 dpi. The amount of the HMW-GS 1Bx14 was also detectably decreased. The percentage of glutenin macropolymeric proteins in total proteins was significantly reduced in the grains of 1Bx14-silenced plants as compared with that in the grains of BSMV:00 infected control plants, indicating that HMW-GS 1Bx14 is one of major components participating in the formation of glutenin macropolymers in wheat grains.
This is one of the first reports of successful application of BSMV-based virus-induced-gene-silencing (VIGS) for gene knockdown in wheat spikes and grains and its application in functional analysis of the 1Bx14 gene. The established BSMV-VIGS system will be very useful in future research on functional analysis of genes contributing to grain quality and the metabolic networks in developing seeds of wheat.
PMCID: PMC3462119  PMID: 22882902
Triticum aestivum; Spike; Grain; Barley stripe mosaic virus (BSMV); Virus-induced gene silencing (VIGS); Functional genomics
8.  CPU-GPU hybrid accelerating the Zuker algorithm for RNA secondary structure prediction applications 
BMC Genomics  2012;13(Suppl 1):S14.
Prediction of ribonucleic acid (RNA) secondary structure remains one of the most important research areas in bioinformatics. The Zuker algorithm is one of the most popular methods of free energy minimization for RNA secondary structure prediction. Thus far, few studies have been reported on the acceleration of the Zuker algorithm on general-purpose processors or on extra accelerators such as Field Programmable Gate-Array (FPGA) and Graphics Processing Units (GPU). To the best of our knowledge, no implementation combines both CPU and extra accelerators, such as GPUs, to accelerate the Zuker algorithm applications.
In this paper, a CPU-GPU hybrid computing system that accelerates Zuker algorithm applications for RNA secondary structure prediction is proposed. The computing tasks are allocated between CPU and GPU for parallel cooperate execution. Performance differences between the CPU and the GPU in the task-allocation scheme are considered to obtain workload balance. To improve the hybrid system performance, the Zuker algorithm is optimally implemented with special methods for CPU and GPU architecture.
Speedup of 15.93× over optimized multi-core SIMD CPU implementation and performance advantage of 16% over optimized GPU implementation are shown in the experimental results. More than 14% of the sequences are executed on CPU in the hybrid system. The system combining CPU and GPU to accelerate the Zuker algorithm is proven to be promising and can be applied to other bioinformatics applications.
PMCID: PMC3303730  PMID: 22369626

Results 1-8 (8)