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BackgroundMesenchymal stromal/stem cells (MSCs) have been intensively investigated in both pre-clinical and clinical studies. However, the therapeutic efficacy varies resulting from the heterogenicity of MSCs. Therefore, purifying the specific MSC subpopulation with specialized function is necessary for their therapeutic applications.MethodsThe large-scale RNA sequencing analysis was performed to identify potential cell markers for the mouse MSCs. Then, the immune suppression activities of the purified MSC subpopulation were assessed in vitro and in vivo.ResultsThe TNFAIP6 (tumor necrosis factor alpha-induced protein 6) has been identified as a potential cell marker for mouse MSCs, irrespective of tissue origin and laboratory origin. The TNFAIP6+mouse MSCs showed enhanced immune suppression activities and improved therapeutic effects on the mouse model of acute inflammation, resulting from faster response to immune stimulation.ConclusionsTherefore, we have demonstrated that the TNFAIP6+MSC subpopulation has enhanced immune suppression capabilities.

Objectives Myocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches. Materials and Methods Four transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations. Results The self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach. Conclusion Thus, the optimized strategy for iCS generation is safer and has more therapeutic potentials., Previously, we and others have successfully reprogrammed mouse fibroblast cells into cardiac progenitor‐like cells. However, several issues should be further addressed, including the reprogramming strategy and delivery approach for myocardial infarction (MI) treatment. In the current study, we screened several transgene‐free approaches for somatic reprogramming. Our data showed the self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. Then, the CXCR4+ (C‐X‐C chemokine receptor 4) subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a more safer delivery approach. Thus, the optimized strategy for iCS generation is much safer and has more therapeutic potentials.

Mesenchymal stem cells (MSCs) can be derived from various adult tissues with multipotent and self‐renewal abilities. The characteristics of presenting no major ethical concerns, having low immunogenicity and possessing immune modulation functions make MSCs promising candidates for stem cell therapies. MSCs could promote inflammation when the immune system is underactivated and restrain inflammation when the immune system is overactivated to avoid self‐overattack. These cells express many immune suppressors to switch them from a pro‐inflammatory phenotype to an anti‐inflammatory phenotype, resulting in immune effector cell suppression and immune suppressor cell activation. We would discuss the mechanisms governing the immune modulation function of these cells in this review, especially the immune‐suppressive effects of MSCs.

Background Although mesenchymal stem cells (MSCs) might offer a promising strategy for treating SLE, their immunoregulatory plasticity makes their therapeutic effects unpredictable. Whether overexpressing IL-37, an IL-1 family member with immunosuppressive activity, might enhance the therapeutic effects of these cells for SLE is unknown. Methods We genetically modified MSCs to overexpress IL-37 and assessed their effects on immune suppression in vitro. We also evaluated the effects of such cells versus effects of various controls after transplanting them into MRL/lpr mice (model of SLE). Results Stem cell characteristics did not appear altered in MSCs overexpressing IL-37. These cells had enhanced immunosuppression in vitro in terms of inhibiting splenocyte proliferation, reducing proinflammatory factors (IL-1β, TNF-α, IL-17, and IL-6), and suppressing autoantibodies (anti-dsDNA and anti-ANA). Compared with animals receiving control MSCs or IL-37 treatment alone, MRL/lpr mice transplanted with IL-37-overexpressing cells displayed improved survival and reduced signs of SLE (indicated by urine protein levels, spleen weight, and renal pathologic scores); they also had significantly lower expression of proinflammatory factors, lower total antibody levels in serum and urine, lower autoantibody production, and showed reduced T cell numbers in the serum and kidney. Expression of IL-37 by MSCs can maintain higher serum levels of IL-37, and MSCs had prolonged survival after transplantation, perhaps through IL-37 suppressing the inflammatory microenvironment. Conclusions Mutually reinforcing interaction between MSCs and IL-37 appears to underlie their additive therapeutic effects. Genetic modification to overexpress IL-37 might offer a way to enhance the stability and effectiveness of MSCs in treating SLE.

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations in lamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases. © Ho et al., published_or_final_version

Cardiospheres represent a more effective cell-based therapy for treatment of myocardial infarction than stem cells of non-cardiac origin. Unfortunately, their therapeutic application is limited by low yield of cell harvesting, declining quality and quantity during the aging process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating cardiosphere-like stem cells from somatic cells via somatic reprogramming. This novel approach would provide an unlimited source of stem cells with cardiac differentiation potential. Here we provide the detailed protocol for generating induced cardiospheres (iCS) for cardiac regeneration by somatic reprogramming of mouse fibroblasts using a panel of pluripotent transcription factors and cardiotrophic growth factors. © 2018 by John Wiley & Sons, Inc.

Nramp (natural resistance associated macrophage protein) has been identified as one of the major candidate genes for controlling natural resistance and/or susceptibility to intracellular pathogens in vertebrates. However, few reports are available about the structure and function of Nramp in teleost. We have recently isolated the cDNA encoding Nramp from turbot (Scophthalmus maximus). The full-length cDNA of the Nramp is 2584 bp in length, including 69 bp 5' terminal UTR, 850 bp 3' terminal UTR and 1665 bp open reading frame for a protein with 554 amino acid residues (Genbank accession number: DQ263240). Comparison of amino acid sequence indicated that turbot Nramp consists of 12 transmembrane regions (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of turbot Nramp exhibited between 60 and 92% homology with 13 other vertebrate Nramp sequences. Nramp transcripts were found to be highly abundant in head kidney, kidney and spleen, abundant in intestine and gill, less abundant in liver, brain, heart and gonad, least in muscle and skin. The level of Nramp mRNA in embryos gradually increases during embryogenesis from blastula stage to fry stage. Challenge of turbot with pathogenic bacteria, Vibrio anguillarum, elevated Nramp mRNA levels in liver and spleen. The Nramp transcripts were detected in turbot embryonic cell line (TEC). Challenge of the TEC cell cultures with pathogenic bacteria, V. anguillarum, significantly elevated Nramp mRNA levels in TEC cell cultures.

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.

Cardiovascular disease is the leading cause of death worldwide. Despite significant progress in understanding of the disease mechanisms, most therapies remain at best palliative. Few therapeutic approaches offer direct tissue repair and regeneration. Cell-based therapy offers a promising approach that involves transplantation of healthy and functional cells to replenish damaged cells and repair injured tissue. Endothelial dysfunction is one of the most important mechanisms of cardiovascular disease, thus endothelial progenitor cells (EPC) and their derivatives have been investigated as a potential source for cell therapy. In pre-clinical and pilot clinical studies, treatment with EPCs or their derivatives as well as their co-transplantation with other cell types has shown some initial promising results. In this review, we will first describe the importance of endothelial cells and EPC homeostasis in the pathophysiology of cardiovascular disease. The potential sources of EPCs, including their isolation and purification, differentiation from pluripotent stem cells and adult stem cells, and trans-differentiation from somatic cells will then be summarized. Lastly, the application of target genome editing tools, such as Zinc Finger Nuclease (ZFN), Transcription Activator Like Effector Nucleases (TALEN) and RNA Guided Endo Nuclease (RGEN) to modify EPCs and their derivatives will be described. These technologies promise to further improve the therapeutic potential of EPCs and their derivatives to treat cardiovascular disease.

Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches.

The potential of human embryonic stem cells (ESCs) for regenerative medicine is unquestionable, but practical and ethical considerations have hampered clinical application and research. In an attempt to overcome these issues, the conversion of somatic cells into pluripotent stem cells similar to ESCs, commonly termed nuclear reprogramming, has been a top objective of contemporary biology. More than 40 years ago, King, Briggs, and Gurdon pioneered somatic cell nuclear reprogramming in frogs, and in 1981 Evans successfully isolated mouse ESCs. In 1997 Wilmut and collaborators produced the first cloned mammal using nuclear transfer, and then Thomson obtained human ESCs from in vitro fertilized blastocysts in 1998. Over the last 2 decades we have also seen remarkable findings regarding how ESC behavior is controlled, the importance of which should not be underestimated. This knowledge allowed the laboratory of Shinya Yamanaka to overcome brilliantly conceptual and technical barriers in 2006 and generate induced pluripotent stem cells (iPSCs) from mouse fibroblasts by overexpressing defined combinations of ESC-enriched transcription factors. Here, we discuss some important implications of human iPSCs for biology and medicine and also point to possible future directions.

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1-60, TRA-1-80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions.

SummarySomatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. However, the low efficiency and slow kinetics of the reprogramming process have hampered progress with this technology. Here we report that a natural compound, vitamin C (Vc), enhances iPSC generation from both mouse and human somatic cells. Vc acts at least in part by alleviating cell senescence, a recently identified roadblock for reprogramming. In addition, Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state. Our results therefore highlight a straightforward method for improving the speed and efficiency of iPSC generation and provide additional insights into the mechanistic basis of the reprogramming process.

Half-smooth tongue sole (Cynoglossus semilaevis) is an increasingly important aquaculture species in China. It is also a tractable model to study sex chromosome evolution and to further elucidate the mechanism of sex determination in teleosts. Two bacterial artificial chromosome (BAC) libraries for C. semilaevis, with large, high-quality inserts and deep coverage, were constructed in the BamHI and HindIII sites of the vector pECBAC1. The two libraries contain a total of 55,296 BAC clones arrayed in 144 384-well microtiter plates and correspond to 13.36 haploid genome equivalents. The combined libraries have a greater than 99% probability of containing any single-copy sequence. Screening high-density arrays of the libraries with probes for female-specific markers and sex-related genes generated between 4-46 primary positive clones per probe. Thus, the two BAC libraries of C. semilaevis provided a readily useable platform for genomics research, illustrated by the isolation of sex determination gene(s).

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be > or =30 mJ/cm(2). The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20-25 min at 5 degrees C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.

The cDNA of myogenic factor (Myf5) was isolated from sea perch (Lateolabrax japonicus) using Reverse-transcription Polymerase Chain Reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 5' flanking sequence of the cDNA contains a TATA box, GC box, CAAT box, several E box sites and muscle-specific regulatory elements determined by genome walking. The Myf5 gene consists of 3 exons and 2 introns. The open reading frame was found to code a protein with 238 amino-acid residues, containing the conserved basic helix-loop-helix domain (bHLH). RT-PCR indicated the Myf5 was highly expressed in muscle, and weakly expressed in brain, eyes, spleen, gill, liver, kidney, intestine and heart. In early embryonic stages, Myf5 mRNA transcripts are highly detectable in the early gastrula stage while decreasing up to a low level at the late gastrula stage, subsequently greatly increased up to the highest level in the somites stage, then gradually decreases from the tail-bud stage to 15 d larvae after hatching, but they are still detectable. Further, Myf5 mRNA was expressed in several sea perch cell lines such as LJES1, LJHK, LJH-1, LJH-2, LJS, LJL, although its expression level varied greatly among different cell lines.

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP