Assessment of the feasibility of human amniotic membrane stem cell-derived cardiomyocytes in vitro.

Myocardial infarction (MI) is a leading cause of death worldwide, resulting in extensive loss of cardiomyocytes and subsequent heart failure. Inducing cardiac differentiation of stem cells is a potential approach for myocardial regeneration therapy to improve post-MI prognosis. Mesenchymal stem cells (MSCs) have several advantages, including immune privilege and multipotent differentiation potential. This study aimed to explore the feasibility of chemically inducing human amniotic membrane MSCs (hAMSCs) to differentiate into cardiomyocytes in vitro. Human amniotic membrane (AM) samples were obtained from routine cesarean sections at Far Eastern Memorial Hospital. The isolated cells exhibited spindle-shaped morphology and expressed surface antigens CD73, CD90, CD105, and CD44, while lacking expression of CD19, CD11b, CD19, CD45, and HLA-DR. The SSEA-1, SSEA-3, and SSEA-4 markers were also positive, and the cells displayed the ability for tri-lineage differentiation into adipocytes, chondrocytes, and osteoblasts. The expression levels of MLC2v, Nkx2.5, and MyoD were analyzed using qPCR after applying various protocols for chemical induction, including BMP4, ActivinA, 5-azacytidine, CHIR99021, and IWP2 on hAMSCs. The group treated with 5 ng/ml BMP4, 10 ng/ml Activin A, 10 µM 5-azacytidine, 7.5 µM CHIR99021, and 5 µM IWP 2 expressed the highest levels of these genes. Furthermore, immunofluorescence staining demonstrated the expression of α-actinin and Troponin T in this group. In conclusion, this study demonstrated that hAMSCs can be chemically induced to differentiate into cardiomyocyte-like cells in vitro. However, to improve the functionality of the differentiated cells, further investigation of inductive protocols and regimens is needed.

Oxidative Stress-Induced Alterations of Cellular Localization and Expression of Aquaporin 1 Lead to Defected Water Transport upon Peritoneal Fibrosis.

Being one of the renal replacement therapies, peritoneal dialysis (PD) maintains around 15% of end-stage kidney disease patients' lives; however, complications such as peritoneal fibrosis and ultrafiltration failure during long-term PD compromise its application. Previously, we established a sodium hypochlorite (NaClO)-induced peritoneal fibrosis porcine model, which helped to bridge the rodent model toward pre-clinical human peritoneal fibrosis research. In this study, the peritoneal equilibration test (PET) was established to evaluate instant functional changes in the peritoneum in the pig model. Similar to observations from long-term PD patients, increasing small solutes transport and loss of sodium sieving were observed. Mechanistic investigation from both in vivo and in vitro data suggested that disruption of cytoskeleton induced by excessive reactive oxygen species defected intracellular transport of aquaporin 1, this likely resulted in the disappearance of sodium sieving upon PET. Functional interference of aquaporin 1 on free water transport would result in PD failure in patients.

S100A8/A9 Enhances Immunomodulatory and Tissue-Repairing Properties of Human Amniotic Mesenchymal Stem Cells in Myocardial Ischemia-Reperfusion Injury.

Paracrine factors of human mesenchymal stem cells (hMSCs) have the potential of preventing adverse cardiac remodeling after myocardial infarction (MI). S100A8 and S100A9 are calcium-binding proteins playing essential roles in the regulation of inflammation and fibrous tissue formation, and they might modulate the paracrine effect of hMSCs. We isolated human amniotic mesenchymal stem cells (hAMSCs) and examined the changes in the expression level of regulatory genes of inflammation and fibrosis after hAMSCs were treated with S100A8/A9. The anti-inflammatory and anti-fibrotic effects of hAMSCs pretreated with S100A8/A9 were shown to be superior to those of hAMSCs without S100A8/A9 pretreatment in the cardiomyocyte hypoxia/reoxygenation experiment. We established a murine myocardial ischemia/reperfusion model to compare the therapeutic effects of the conditioned medium of hAMSCs with or without S100A8/A9 pretreatment. We found the hearts administered with a conditioned medium of hAMSCs with S100A8/A9 pretreatment had better left ventricular systolic function on day 7, 14, and 28 after MI. These results suggest S100A8/A9 enhances the paracrine therapeutic effects of hAMSCs in aspects of anti-inflammation, anti-fibrosis, and cardiac function preservation after MI.

New design to remove leukocytes from platelet-rich plasma (PRP) based on cell dimension rather than density.

Platelet-rich plasma (PRP) can stimulate the proliferation of stem cells and have a positive effect on tissue repair. Although many commercialized PRP preparation kits are already on the market, first-line clinical workers are still not satisfied with most of the PRP kits. The work of commercial PRP kits is based on the density of blood elements. However, the blood elements are very close in density which makes the separation challenging. Therefore, the mentioned commercialized kits are generally contaminated by leucocytes and erythrocyte. In this study, a home-designed PRP device was developed to use a separation membrane with adequate cut-off pore size of 5 µm, 3 µm and 2 µm for the groups of H5M, H3M, and H2M, respectively, to be placed in the middle of the centrifuge tube. The home-designed H2M showed a very promising results regardless of the final volume (1.82 ± 0.09 ml), platelet yield (8.39 ± 0.44%), Red Blood Cells (0%), White Blood Cells (0%), and Relative Concentration of Platelet Increment value (225.09%). Further, it showed a good result in cell viability and cytotoxicity and confirmed a good multilineage potentials. The concentration in PRP prepared by group H2M was relatively stable and far above average. All the fibrin fibers were linked together as bridging strands or strings and turned into an inter-connected porous structure for nutrients transportation and regenerative cell migration. We believe that the home-designed group H2M should have a great potential to develop into the final product to meet the requirements of first-line clinical workers.

Overexpression of Adiponectin Receptor 1 Inhibits Brown and Beige Adipose Tissue Activity in Mice.

Adult humans and mice possess significant classical brown adipose tissues (BAT) and, upon cold-induction, acquire brown-like adipocytes in certain depots of white adipose tissues (WAT), known as beige adipose tissues or WAT browning/beiging. Activating thermogenic classical BAT or WAT beiging to generate heat limits diet-induced obesity or type-2 diabetes in mice. Adiponectin is a beneficial adipokine resisting diabetes, and causing "healthy obese" by increasing WAT expansion to limit lipotoxicity in other metabolic tissues during high-fat feeding. However, the role of its receptors, especially adiponectin receptor 1 (AdipoR1), on cold-induced thermogenesis in vivo in BAT and in WAT beiging is still elusive. Here, we established a cold-induction procedure in transgenic mice over-expressing AdipoR1 and applied a live 3-D [18F] fluorodeoxyglucose-PET/CT (18F-FDG PET/CT) scanning to measure BAT activity by determining glucose uptake in cold-acclimated transgenic mice. Results showed that cold-acclimated mice over-expressing AdipoR1 had diminished cold-induced glucose uptake, enlarged adipocyte size in BAT and in browned WAT, and reduced surface BAT/body temperature in vivo. Furthermore, decreased gene expression, related to thermogenic Ucp1, BAT-specific markers, BAT-enriched mitochondrial markers, lipolysis and fatty acid oxidation, and increased expression of whitening genes in BAT or in browned subcutaneous inguinal WAT of AdipoR1 mice are congruent with results of PET/CT scanning and surface body temperature in vivo. Moreover, differentiated brown-like beige adipocytes isolated from pre-adipocytes in subcutaneous WAT of transgenic AdipoR1 mice also had similar effects of lowered expression of thermogenic Ucp1, BAT selective markers, and BAT mitochondrial markers. Therefore, this study combines in vitro and in vivo results with live 3-D scanning and reveals one of the many facets of the adiponectin receptors in regulating energy homeostasis, especially in the involvement of cold-induced thermogenesis.

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (-1369~+28 nt), Δ2-pGzmg (-939~+28 nt), Δ3-pGzmg (-711~+28 nt) and Δ4-pGzmg (-417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the -417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

The development of laminin-alginate microspheres encapsulated with Ginsenoside Rg1 and ADSCs for breast reconstruction after lumpectomy.

Many technologies have been developed for breast reconstruction after lumpectomy. Although the technologies achieved promising success in clinical, there are still many shortages hanging over and trouble the researchers. Tissue engineering technology was introduced to plastic surgery that gave a light to lumpectomy patients in breast reconstruction. The unexpected absorption rate, resulting from limited vascularization and low cell survival rate, is a major factor that leads to unsatisfactory results for the previous studies in our lab. In the study, the laminin-modified alginate synthesized by a new method of low concertation of sodium periodate would be mixed with ADSCs and Rg1 in the medium; and then sprayed into a calcium chloride (CaCl2) solution to prepare into microsphere (abbreviated as ADSC-G-LAMS) by bio-electrospray with a power syringe for the mass production and smaller bead size. The developed ADSC-G-LAMS microspheres had the diameter of 232 ± 42 µm. Sustained-release of the Rg1 retained its biological activity. WST-1, live/dead staining, and chromosome aberration assay were evaluated to confirm the safety of the microspheres. In in vivo study, ADSC-G-LAMS microspheres combined with autologous adipocytes were transplanted into the dorsum of rats by subcutaneous injection. The efficacy was investigated by H&E and immunofluorescence staining. The results showed that the bioactive ADSC-G-LAMS microspheres could integrate well into the host adipose tissue with an adequate rate of angiogenesis by constantly releasing Rg1 to enhance the ADSC or adipocyte survival rate to join tissue growth and repair with adipogenesis for breast reconstruction after lumpectomy.

Glucocorticoid transiently upregulates mitochondrial biogenesis in the osteoblast.

Glucocorticoid (GC)-induced bone loss is the most prevalent form of secondary osteoporosis. Previous studies demonstrated that long-term incubation of dexamethasone (DEX) induced oxidative stress and mitochondrial dysfunctions, consequently leading to apoptosis of differentiated osteoblasts. This DEX-induced cell death might be the main causes of bone loss. We previously described that DEX induced biphasic mitochondrial alternations. As GC affects mitochondrial physiology through several different possible routes, the short-term and long-term effects of GC treatment on mitochondria in the osteoblast have not been carefully characterized. Here, we examined the expression levels of genes that are associated with mitochondrial functions at several different time points after incubation with DEX. Mitochondrial biogenesis-mediated genes nuclear respiratory factor 1 (Nrf1) and Nrf2 were upregulated after 4-h incubation, and then declined after 24-h incubation, suggesting that mitochondrial biogenesis were transiently upregulated by DEX. In contrast, mitochondrial fusion gene optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2) started to be elevated as the biogenesis started to decrease. Finally, the mitochondrial fission increased and apoptosis becomes prominent. Agree with the mitochondrial biphasic alterations hypothesis, the results suggested an early increase of mitochondrial activities and biogenesis upon DEX stimulation to the osteoblasts. The oxidative phosphorylation and inducible nitric oxide synthase levels increased results in oxidative stress accumulation, leading to mitochondrial fusion, and subsequently fission and triggering the apoptosis. Our results indicated that the primary effects of GC on mitochondria are promoting their functions and biogenesis. Mitochondrial breakdown and the activation of the apoptotic pathways appeared to be the secondary effect after long-term treatment.

Olfactory ensheathing cells improve the survival of porcine neural xenografts in a Parkinsonian rat model.

BACKGROUND: Parkinson's disease (PD) features the motor control deficits resulting from irreversible, progressive degeneration of dopaminergic (DA) neurons of the nigrostriatal pathway. Although intracerebral transplantation of human fetal ventral mesencephalon (hfVM) has been proven effective at reviving DA function in the PD patients, this treatment is clinically limited by availability of hfVM and the related ethical issues. Homologous tissues to hfVM, such as porcine fetal ventral mesencephalon (pfVM) thus present a strong clinical potential if immune response following xenotransplantation could be tamed. Olfactory ensheathing cells (OECs) are glial cells showing immunomodulatory properties. It is unclear but intriuging whether these properties can be applied to reducing immune response following neural xenotransplantation of PD. METHODS: To determine whether OECs may benefit neural xenografts for PD, different compositions of grafting cells were transplanted into striatum of the PD model rats. We used apomorphine-induced rotational behavior to evaluate effectiveness of the neural grafts on reviving DA function. Immunohistochemistry was applied to investigate the effect of OECs on the survival of neuroxenografts and underlying mechanisms of this effect. RESULTS: Four weeks following the xenotransplantation, we found that the PD rats receiving pfVM + OECs co-graft exhibited a better improvement in apomorphine-induced rotational behavior compared with those receiving only pfVM cells. This result can be explained by higher survival of DA neurons (tyrosine hydroxylase immunoreactivity) in grafted striatum of pfVM + OECs group. Furthermore, pfVM + OECs group has less immune response (CD3+ T cells and OX-6+ microglia) around the grafted area compared with pfVM only group. These results suggest that OECs may enhance the survival of the striatal xenografts via dampening the immune response at the grafted sites. CONCLUSIONS: Using allogeneic OECs as a co-graft material for xenogeneic neural grafts could be a feasible therapeutic strategy to enhance results and applicability of the cell replacement therapy for PD.

The Effect of Sertoli Cells on Xenotransplantation and Allotransplantation of Ventral Mesencephalic Tissue in a Rat Model of Parkinson's Disease.

Intra-striatal transplantation of fetal ventral mesencephalic (VM) tissue has a therapeutic effect on patients with Parkinson's disease (PD). Sertoli cells (SCs) possess immune-modulatory properties that benefit transplantation. We hypothesized that co-graft of SCs with VM tissue can attenuate rejection. Hemi-parkinsonian rats were generated by injecting 6-hydroxydopamine into the right medial forebrain bundle of Sprague Dawley (SD) rats. The rats were then intrastriatally transplanted with VM tissue from rats or pigs (rVM or pVM), with/without a co-graft of SCs (rVM+SCs or pVM+SCs). Recovery of dopaminergic function and survival of the grafts were evaluated using the apomorphine-induced rotation test and small animal-positron emission tomography (PET) coupled with [18F] DOPA or [18F] FE-PE2I, respectively. Immunohistochemistry (IHC) examination was used to determine the survival of the grafted dopaminergic neurons in the striatum and to investigate immune-modulatory effects of SCs. The results showed that the rVM+SCs and pVM+SCs groups had significantly improved drug-induced rotational behavior compared with the VM alone groups. PET revealed a significant increase in specific uptake ratios (SURs) of [18F] DOPA and [18F] FE-PE2I in the grafted striatum of the rVM+SCs and pVM+SCs groups as compared to that of the rVM and pVM groups. SC and VM tissue co-graft led to better dopaminergic (DA) cell survival. The co-grafted groups exhibited lower populations of T-cells and activated microglia compared to the groups without SCs. Our results suggest that co-graft of SCs benefit both xeno- and allo-transplantation of VM tissue in a PD rat model. Use of SCs enhanced the survival of the grafted dopaminergic neurons and improved functional recovery. The enhancement may in part be attributable to the immune-modulatory properties of SCs. In addition, [18F]DOPA and [18F]FE-PE2I coupled with PET may provide a feasible method for in vivo evaluation of the functional integrity of the grafted DA cell in parkinsonian rats.

Bone regeneration in Ds-Red pig calvarial defect using allogenic transplantation of EGFP-pMSCs - A comparison of host cells and seeding cells in the scaffold.

BACKGROUND: Cells, scaffolds, and factors are the triad of regenerative engineering; however, it is difficult to distinguish whether cells in the regenerative construct are from the seeded cells or host cells via the host blood supply. We performed a novel in vivo study to transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial defect of DsRed pigs. The cell distribution and proportion were distinguished by the different fluorescent colors through the whole regenerative period. METHOD/RESULTS: Eight adult domestic Ds-Red pigs were treated with five modalities: empty defects without scaffold (group 1); defects filled only with scaffold (group 2); defects filled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 x 103 cells/scaffold (group 4); and defects filled with 5 x 104 cells/scaffold (group 5). The in vitro cell distribution, morphology, osteogenic differentiation, and fluorescence images of groups 4 and 5 were analyzed. Two animals were sacrificed at 1, 2, 3, and 4 weeks after transplantation. The in vivo fluorescence imaging and quantification data showed that EGFP-pMSCs were represented in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded cell density. Host cells were recruited by seeded cells if enough space was available in the scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which indicates that the scaffold without seeded cells cannot recruit host cells even when enough space is available for cell ingrowth. The histological and immunohistochemical data showed that more cells were involved in osteogenesis in scaffolds with seeded cells. CONCLUSION: Our in vivo results showed that more seeded cells recruit more host cells and that both cell types participate in osteogenesis. These results suggest that scaffolds without seeded cells may not be effective in bone transplantation.

Different susceptibilities of osteoclasts and osteoblasts to glucocorticoid-induced oxidative stress and mitochondrial alterations.

Glucocorticoid-induced bone loss is the most common form of secondary osteoporosis. This toxic effect has not been efficiently managed, possibly due to the incomplete understanding of the extraordinarily diverse cellular responses induced by glucocorticoid treatment. Previous literatures revealed that high dose of exogenous glucocorticoid triggers apoptosis in osteocytes and osteoblasts. This cell death is associated with glucocorticoid-induced oxidative stress. In this study, we aimed to investigate the mechanisms of glucocorticoid-induced apoptosis in osteoblasts and examine the responses of osteoclasts to the synthetic glucocorticoid, dexamethasone. We demonstrated the biphasic effects of exogenous glucocorticoid on osteoblastic mitochondrial functions and elevated intracellular oxidative stress in a dose- and time-dependent manner. On comparison, similar treatment did not induce mitochondrial dysfunctions and oxidative stress in osteoclasts. The production of reactive oxygen/nitrogen species was decreased in osteoclasts. The differences are not due to varying efficiency of cellular antioxidant system. The opposite effects on nitrogen oxide synthase might provide an explanation, as the expression levels of nos2 gene are suppressed in the osteoclast but elevated in the osteoblast. We further revealed that glucocorticoids have a substantial impact on the osteoblastic mitochondria. Basal respiration rate and ATP production were increased upon 24 h incubation of glucocorticoids. The increase in proton leak and nonmitochondrial respiration suggests a potential source of glucocorticoid-induced oxidative stress. Long-term incubation of glucocorticoids accumulates these detrimental changes and results in cytochrome C release and mitochondrial breakdown, consequently leading to apoptosis in osteoblasts. The mitochondrial alterations might be other sources of glucocorticoid-induced oxidative stress in osteoblasts.

A mini-pig model for evaluating the efficacy of autologous platelet patches on induced acute full thickness wound healing.

BACKGROUND: Autologous platelet concentrates are currently widely used across different areas of regenerative medicine in order to enhance the wound healing process. Although several protocols for platelet concentrates are available, their application remains difficult due to different protocols leading to distinct products with vary potential biological uses. In this study, we attempted to make a platelet patch (PP) using mixtures of platelet rich plasma (PRP) injection and platelet rich fibrin (PRF) to promote wound repair and regeneration. RESULTS: Experiments were performed using a full-thickness wound model in mini-pigs. Autologous PRP, PRF and PP were prepared immediately before creating four full-thickness skin wounds in pigs. We quantified concentrations of platelets, thrombin and various growth factors to ensure that the desired effect can be produced. After surgery, hydrocolloid dressing, PRP injection, PRF and PP was applied to experimentally induced wounds. Application efficacy was evaluated by measurement of wound sizes and histological examination. The results indicated that all wounds showed a significant size reduction. Wound repair efficacy in response to PP treatment exhibited enhanced re-epithelialization compared to PRP and PRF (P < 0.05) and higher wound contraction than did PRF application (P < 0.05). Another aspect, experiment using DsRed transgenic pigs as blood donors demonstrated that leucocytes in PP were incorporated into the wound bed at the end of the study, suggesting that leucocytes activity is stimulated in response to PP application. Safety of the experimental processes was also confirmed by examination of organ biopsies. CONCLUSIONS: We used a mini-pig model to evaluate the efficacy of lab-made PP on induced full-thickness wound healing. Results demonstrated that application of one piece of PP was enough to obtain comparable efficacy versus general utilization of PRP or PRF for wound care. We also demonstrated that leucocytes in PP were incorporated into the wound bed and no safety concerns have been found in the whole experiment. This study provides a novel and feasible method for veterinary or clinical wound care.

In vitro culture and characterization of duck primordial germ cells.

This study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Three-dimensional culture of chicken primordial germ cells (cPGCs) in defined media containing the functional polymer FP003.

Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.

BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model.

Gene therapy for bone tissue engineering has been widely developed. Recently, non-viral DNA-based gene therapy has been reported to be a safer and more efficient method of delivering DNA into target cells. We used a non-viral gene transfection reagent to delivery bone morphogenetic protein-2 (BMP-2) gene into bone marrow stromal cells (BMSCs). Primary BMSCs were isolated from rat femurs and transfected with BMP-2 plasmids. The transfection rate was analyzed using flow cytometry. The concentration of BMP-2 protein was quantified using an enzyme-linked immunosorbent assay. Levels of osteopontin and osteocalcin were measured to evaluate osteogenic differentiation. In vivo, we designed a critical-size calvarial defect rat model to study new bone regeneration, using Matrigel as a scaffold to carry BMP-2-transfected bone marrow stromal cells into the defect site. New bone formation was assessed by micro-computed tomography, X-ray, immunohistochemical staining and histomophometry. The transfection rate after 72 h was 31.5%. The BMP-2 protein level as well as osteopontin and osteocalcin expressions were higher in the experimental group (transfected with BMP-2) than the control group (transfected with green fluorescent protein, GFP). The in vivo study suggested that bone healing occurred 12 weeks after scaffold implantation. In addition, BMP-2-transfected bone marrow stromal cells provided better osteogenic differentiation than primary bone marrow stromal cells. Our findings suggest that non-viral gene therapy may be useful in bone tissue engineering. SIGNIFICANCE: The study has clinical implications for the wider use of BMP-2-transfected BMSCs for cell-based transplantation therapy in bone regeneration.

Correction to: Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

Following publication of the original article [1], the authors reported that one of the authors' names is spelled incorrectly.

Stage-dependent piRNAs in chicken implicated roles in modulating male germ cell development.

BACKGROUND: The PIWI/piRNA pathway is a conserved machinery important for germ cell development and fertility. This piRNA-guided molecular machinery is best known for repressing derepressed transposable elements (TE) during epigenomic reprogramming. The extent to which piRNAs are involved in modulating transcripts beyond TEs still need to be clarified, and it may be a stage-dependent event. We chose chicken germline as a study model because of the significantly lower TE complexity in the chicken genome compared to mammalian species. RESULTS: We generated high-confidence piRNA candidates in various stages across chicken germline development by 3'-end-methylation-enriched small RNA sequencing and in-house bioinformatics analysis. We observed a significant developmental stage-dependent loss of TE association and a shifting of the ping-pong cycle signatures. Moreover, the stage-dependent reciprocal abundance of LINE retrotransposons, CR1-C, and its associated piRNAs implicated the developmental stage-dependent role of piRNA machinery. The stage dependency of piRNA expression and its potential functions can be better addressed by analyzing the piRNA precursors/clusters. Interestingly, the new piRNA clusters identified from embryonic chicken testes revealed evolutionary conservation between chickens and mammals, which was previously thought to not exist. CONCLUSIONS: In this report, we provided an original chicken RNA resource and proposed an analytical methodology that can be used to investigate stage-dependent changes in piRNA compositions and their potential roles in TE regulation and beyond, and also revealed possible conserved functions of piRNAs in developing germ cells.

Surface modification: activation and deactivation of osteogenic differentiation based on detachable growth factor protein.

An advanced material interface is modified by using a substrate-independent coating of detachable poly-para-xylylene, enabling dynamical control of the immobilization and detachment of biomolecules, and a previously installed biological function is deactivated or tuned with reduced activity. The induction of osteogenesis activity, and subsequent deactivation of such osteogenesis activity, is demonstrated.

Infusion of Porcine-Derived Amniotic Fluid Stem Cells for Treatment of Experimental Colitis in Mice.

Recently, stem cells have offered an alternative treatment for inflammatory bowel disease (IBD) or colitis to overcome the poor outcomes associated with current therapies. Amniotic fluid-derived stem cells (AFSCs) have the potential for the regeneration of impaired organs and the recovery of normal physiologic functions of damaged tissues without ethical concerns or risk of tumor formation. In this work, we aimed to examine the therapeutic effects of infusion of porcine AFSCs (pAFSCs) in dextran sulfate sodium (DSS)-induced colitis in mice. Treatment with pAFSCs was shown to inhibit the shortening of the colon after induction of colitis and dramatically ameliorated the body weightloss induced by the DSS treatment. In addition, pAFSCs could also reduce the extent of the inflamed area represented by epithelial mesenchymal transformation in the colitis mice. The levels of the inflammatory cytokines interleukin 6 (IL-6) and interferon gamma (IFN-γ) were also reduced in colitis mice transplanted with pAFSCs. In conclusion, pAFSCs can ameliorate experimental colitis in mice, suggesting that they may be a potential treatment for IBD or colitis.