Rat iPSCs Research Articles

Generation of induced pluripotent stem cells from rat fibroblasts and optimization of its differentiation into mature functional neurons

BackgroundInduced pluripotent stem cells (iPSCs) derived neural stem cells (NSCs) provide a potential for autologous neural transplantation therapy following neurological insults. Thus far, in preclinical studies the donor iPSCs-NSCs are mostly of human or mouse origin with concerns centering around graft rejection when applied to rat brain injury models. For better survival and integration of transplanted cells in the injured brain in rat models, use of rat-iPSC-NSCs and in combination with biomaterials is of advantageous. Herein, we report a detailed method in generating rat iPSCs with improved reprogramming efficiency and differentiation into neurons. New methodRat fibroblasts were reprogrammed into iPSCs with polybrene and EF1α-STEMCCA-LoxP lentivirus vector. Pluripotency characterization, differentiation into neuronal linage cells were assessed with RT-qPCR, Western blotting, immunostaining and patch-clamp methods. Cells were cultured in a custom-designed integrin array system as well as in a hydrogel-based 3D condition. ResultsWe describe a thorough method for the generation of rat-iPSC-NSCs, and identify integrin αvβ8 as a substrate for the optimal growth of rat-iPSC-NSCs. Furthermore, with hydrogel as the supporting biomaterial in the 3-D culture, when combined with integrin αvβ8 binding peptide, it forms a conducive environment for optimal growth and differentiation of iPSC-NSCs into mature neurons. Comparison with existing methodsPublished studies about rat-iPSC-NSCs are rare. This study provides a detailed protocol for the generation of rat iPSC-NSCs and optimal growth conditions for neuronal differentiation. Our method is useable for studies to assess the utility of rat iPSC-NSCs for neural transplantation in rat brain injury models.

Siponimod Attenuates Neuronal Cell Death Triggered by Neuroinflammation via NFκB and Mitochondrial Pathways.

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system (CNS), consisting of heterogeneous clinical courses varying from relapsing-remitting MS (RRMS), in which disability is linked to bouts of inflammation, to progressive disease such as primary progressive MS (PPMS) and secondary progressive MS (SPMS), in which neurological disability is thought to be linked to neurodegeneration. As a result, successful therapeutics for progressive MS likely need to have both anti-inflammatory and direct neuroprotective properties. The modulation of sphingosine-1-phosphate (S1P) receptors has been implicated in neuroprotection in preclinical animal models. Siponimod/BAF312, the first oral treatment approved for SPMS, may have direct neuroprotective benefits mediated by its activity as a selective (S1P receptor 1) S1P1 and (S1P receptor 5) S1P5 modulator. We showed that S1P1 was mainly present in cortical neurons in lesioned areas of the MS brain. To gain a better understanding of the neuroprotective effects of siponimod in MS, we used both rat neurons and human-induced pluripotent stem cell (iPSC)-derived neurons treated with the neuroinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Cell survival/apoptotic assays using flow cytometry and IncuCyte live cell analyses showed that siponimod decreased TNF-α induced neuronal cell apoptosis in both rat and human iPSCs. Importantly, a transcriptomic analysis revealed that mitochondrial oxidative phosphorylation, NFκB and cytokine signaling pathways contributed to siponimod's neuroprotective effects. Our data suggest that the neuroprotection of siponimod/BAF312 likely involves the relief of oxidative stress in neuronal cells. Further studies are needed to explore the molecular mechanisms of such interactions to determine the relationship between mitochondrial dysfunction and neuroinflammation/neurodegeneration.

Therapeutic function of a novel rat induced pluripotent stem cell line in a 6‑OHDA‑induced rat model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder of the central nervous system that results from the loss of dopaminergic (DA) nigral neurons. Induced pluripotent stem cells (iPSCs) have shown potential for cell transplantation treatment of neurodegenerative disorders. In the present study, the small molecules CHIR99021 and RepSox (CR) significantly facilitated reprogramming and enhanced the efficiency of GFP+/iPS‑like colonies [rat iPSCs induced by OCT3/4, Sox2, Klf4, c‑Myc, Nanog and Lin28 + CR (RiPSCs‑6F/CR)] generation by ~4.0‑fold during lentivirus‑mediated reprogramming of six transcription factors in rat embryonic fibroblasts. The generation of iPSCs was detected by reverse transcription‑PCR, immunofluorescence and western blot analysis. Subsequently, RiPSCs‑6F/CR were stereotactically transplanted into the right medial forebrain bundle (MFB) of 6‑hydroxydopamine‑lesioned rats with PD. The transplanted RiPSCs‑6F/CR survived and functioned in the MFB of rats with PD for ≥20weeks, and significantly improved functional restoration from their PD‑related behavioral defects. Furthermore, the grafted RiPSCs‑6F/CR could migrate and differentiate into various neurocytes invivo, including γ aminobutyric acid‑ergic, DA neurons and glial cells. In conclusion, the present study confirmed that RiPSCs‑6F/CR induced by small molecules could be used as potential donor material for neural grafting to remodel basal ganglia circuitry in neurodegenerative diseases.

Therapeutic function of iPSCs-derived primitive neuroepithelial cells in a rat model of Parkinson's disease

Induced pluripotent stem cells (iPSCs) are a promising unlimited source for cell replacement therapy of neurodegenerative disorders, including Parkinson's disease (PD). In the present study, rat iPSCs-derived primitive neuroepithelial cells (RiPSCs-iNECs) were successfully induced from rat iPSCs (RiPSCs) following two major developmental stages, and could generate neurospheres and differentiated into both neurons and astrocytes in vitro. Then, the RiPSCs-iNECs-GFP+ were unilaterally transplanted into the right substantia nigra (SN) of 6-hydroxydopamine-lesioned rat models of PD. The results demonstrated that the grafted RiPSCs-iNECs could survive in parkinsonian rat brain for at least 150 days, and many of them differentiated into tyrosine hydroxylase (TH)-positive cells. Furthermore, the PD model rats grafted with RiPSCs-iNECs exhibited a significant functional recovery from their parkinsonian behavioral defects. Histological studies showed that RiPSCs-iNECs could differentiate into multiple types of neurons including dopaminergic neurons, GFAP, Pax6, FoxA2 and DAT-positive cells, and induced dopaminergic neurons extended dense neurites into the host striatum. Thus, iPSCs derived primitive neuroepithelial cells could be an attractive candidate as a source of donor material for the treatment of PD, but the molecular mechanism needs further clarification.

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells.

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.

The effect of dual inhibition of Ras-MEK-ERK and GSK3β pathways on development of in vitro cultured rabbit embryos.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3β pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFβ/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFβ/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

Differentiation and characterization of neurons derived from rat iPSCs

BackgroundInduced pluripotent stem cells (iPSCs) may be an advantageous source of neuronal cells to repair damage due to neurological disorders or trauma. Additionally, they are promising candidates to develop models to study underlying mechanisms of neurodegenerative diseases. While successful neural differentiation of iPSCs has been reported in mice, protocols detailing the generation of neural cells from rat iPSCs are relatively limited, and their optimization by manipulating cell culture methods has remained unexplored. New methodHere, we describe and compare the effects of four distinct, commonly used substrates on the neuronal differentiation of rat iPSC (riPSC) derived-neural progenitor cells. Our approach is to use substrate coating as a method to enrich differentiated riPSCs for neuronal subtypes with the desired morphology and maturity. We use a combination of electrophysiology, immunofluorescence staining, and Sholl analysis to characterize the cells generated on each substrate over a nine-day time course. ResultsThe surface coating presented by the cell culture substrate influences the polarity and arborization of differentiating neurons. Polyornithine-laminin coating promoted neuronal arborization and maturation, while Geltrex favored bipolar cells which displayed indicators of functional immaturity. Poly-d-lysine substrate was associated with limited neurite outgrowth and arborization. Gelatin was the least favorable substrate for the growth and differentiation of our cells.Comparison with Existing Method: Rat-derived neural progenitor cells have been previously derived; however, our methods to use substrate coatings to influence morphological and electrical maturity have not been explored previously. ConclusionSubstrate coatings can be selected to enrich differentiated riPSCs for distinctive neuronal morphologies.

VGF nerve growth factor inducible is involved in retinal ganglion cells death induced by optic nerve crush

VGF nerve growth factor inducible (VGF) is a polypeptide that is induced by neurotrophic factors and is involved in neurite growth and neuroprotection. The mRNA of the Vgf gene has been detected in the adult rat retina, however the roles played by VGF in the retina are still undetermined. Thus, the purpose of this study was to determine the effects of VGF on the retinal ganglion cells (RGCs) of mice in the optic nerve crush (ONC) model, rat-derived primary cultured RGCs and human induced pluripotent stem cells (iPSCs)-derived RGCs. The mRNA and protein of Vgf were upregulated after the ONC. Immunostaining showed that the VGF was located in glial cells including Müller glia and astrocytes but not in the retinal neurons and their axons. AQEE-30, a VGF peptide, suppressed the loss of RGCs induced by the ONC, and it increased survival rat-derived RGCs and promoted the outgrowth of neurites of rat and human iPSCs derived RGCs in vitro. These findings indicate that VGF plays important roles in neuronal degeneration and has protective effects against the ONC on RGCs. Thus, VGF should be considered as a treatment of RGCs degeneration.

A naked mole rat iPSC line expressing drug-inducible mouse pluripotency factors developed from embryonic fibroblasts

Naked mole rats (NMRs, Heterocephalus glaber) are long-lived, cancer-resistant rodents. Here, we report the development of an induced pluripotent stem cell (iPSC) line generated from immortalized NMR embryonic fibroblasts transduced with a doxycycline-inducible mouse OSKM polycistronic vector. This iPSC line was shown to express pluripotency-associated markers, form embryoid bodies, differentiate in vitro to the derivatives of three germ layers, and exhibit normal karyotype. The ability of iPSCs to differentiate in vivo was supported by the contribution to interspecific chimera upon injection into mouse blastocysts. This NMR iPSC line may be a useful tool in cancer and aging research.

Generation of a Sprague-Dawley-GFP rat iPS cell line

We generated a rat iPSC line called ATCi-rSD95 from transgenic Sprague-Dawley GFP fetal fibroblasts. Established ATCi-rSD95 cells present a normal karyotype, silencing of the transgenes and express pluripotency-associated markers. Additionally, ATCi-rSD95 cells are able to form teratoma with differentiated cells derived from the three germ-layers that maintain the GFP expression.

Genetic Manipulation by Zinc-Finger Nucleases in Rat-Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have an extensive application in regenerative medicine, pharmaceutical discovery, and basic research. With the recent derivation of rat iPSCs, it is now feasible to apply genetic manipulation in this species. But such tools do not yet exist for many rat strains, especially for disease model rat. The Sprague Dawley (SD) rat is an inbred disease model for hypertension, nephropathy, pulmonary hypertension, depression, and alcohol consumption. In this study, the SD rat iPSCs were generated using lentiviral method. The p53 gene was targeted in rat iPSCs using homologous recombination mediated by P53 zinc-finger nucleases (ZFNs). Our results showed that these rat iPSCs were pluripotent status. P53 gene was targeted successfully with high efficiency by coelectroporating the donor targeting vectors and p53 ZFN vector to these rat iPSCs. Southern blotting analysis confirmed the correct homologous recombination in rat iPSCs. At the same time, our results demonstrated that the P53 dependent function was abolished in p53-targeted iPSCs. This report also demonstrated that iPS technology, combined with homologous recombination mediated by ZFN, was suitable to develop human disease model in rat and other species.

Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells

Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of “footprint-free” (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate “footprint-free” iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of “footprint-free” iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate “footprint-free” iPSCs, which may advance the translation of iPSC technology into the clinical settings.

Transcriptome Characteristics and X-Chromosome Inactivation Status in Cultured Rat Pluripotent Stem Cells.

Rat pluripotent stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) as mouse and human ones have a great potential for studying mammalian early development, disease modeling, and evaluation of regenerative medicine approaches. However, data on pluripotency realization and self-renewal maintenance in rat cells are still very limited, and differentiation protocols of rat ESCs (rESCs) and iPSCs to study development and obtain specific cell types for biomedical applications are poorly developed. In this study, the RNA-Seq technique was first used for detailed transcriptome characterization in rat pluripotent cells. The rESC and iPSC transcriptomes demonstrated a high similarity and were significantly different from those in differentiated cells. Additionally, we have shown that reprogramming of rat somatic cells to a pluripotent state was accompanied by X-chromosome reactivation. There were two active X chromosomes in XX rESCs and iPSCs, which is one of the key attributes of the pluripotent state. Differentiation of both rESCs and iPSCs led to X-chromosome inactivation (XCI). The dynamics of XCI in differentiating rat cells was very similar to that in mice. Two types of facultative heterochromatin described in various mammalian species were revealed on the rat inactive X chromosome. To explore XCI dynamics, we established a new monolayer differentiation protocol for rESCs and iPSCs that may be applied to study different biological processes and optimized for directed derivation of specific cell types.

Hepatic differentiation of rat induced pluripotent stem cells in vitro.

To show the efficient generation of hepatocyte-like cells (HLCs) differentiated from the induced pluripotent stem cells (iPSCs) of rats. Hepatic differentiation was achieved using a three-step protocol with several growth factors. First, rat iPSCs were differentiated into definitive endoderm cells using Activin A and Wnt3a treatment. Then fibroblast growth factor 4 and bone morphogenetic protein 2 were added to the culture medium and used to induce hepatic differentiation. Finally, hepatocyte growth factor, Oncostatin M and dexamethasone were used for hepatic maturation. The liver-related markers and functions of HLCs were assessed at the gene and protein levels. After endodermal induction, the differentiated cells expressed endodermal markers forkhead box protein A2 and SRY-box containing gene 17 at the mRNA and protein levels. After 20 d of culture, the iPSCs were differentiated into HLCs. These differentiated cells expressed hepatic markers including α-fetoprotein, albumin CK8, CK18, CK19, and transcription factor HNF-4α. In addition, the cells expressed functional proteins such as α1-antitrypsin, cytochrome P450 1A2 and CYP 3A4. They acted like healthy hepatic cells, storing glycogen and taking up indocyanine green and low-density lipoproteins. Also, the rates of urea synthesis (20 d 1.202 ± 0.080 mg/dL vs 0 d 0.317 ± 0.021 mg/dL, P < 0.01) and albumin secretion (20 d 1.601 ± 0.102 mg/dL vs 0 d 0.313 ± 0.015 mg/dL, P < 0.01) increased significantly as differentiation progressed. Rat iPSCs can differentiate into HLCs rapidly and efficiently. These differentiated cells may be an attractive resource for treatment of end-stage liver disease.

Generation and Characterization of Rat iPSCs.

The laboratory rat (Rattus norvegicus) is now on the leading edge used as a laboratory model system to study pharmacology, toxicology, immunology, nutrition, behavior, and numerous other topics. Therefore, generation of rat induced pluripotent stem cells (iPSCs) through somatic cells reprogramming is a powerful tool for establishing in vitro disease model, development of new protocols for treatment of different diseases, and creating transgenic rat models. Here, we describe a simple adopted protocol for establishing rat iPSCs from different types of somatic cells including rat primary ear fibroblast (PEF) and primary bone marrow cells (BMC).

Generation of rat induced pluripotent stem cells using a plasmid vector and possible application of a keratan sulfate glycan recognizing antibody in discriminating teratoma formation phenotypes.

Induced pluripotent stem cells (iPSCs) offer an invaluable tool for biological research and regenerative medicine. We report establishment of rat iPSCs (riPSCs) using a plasmid vector encoding four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. Although all riPSC clones were generated and cultured under the same conditions, expressed hallmark pluripotency markers and differentiated successfully in vitro, the expression of a keratan sulfate glycan epitope with unique properties defined by R-10G antibody varied in the riPSC clones. In contrast, tumor rejection antigen (TRA)-1-81 epitope expression was comparable. A clone highly reactive to R-10G antibody formed teratomas in vivo consisting of cells from all three germ layers. However, clones expressing a lower level of the epitope defined by R-10G resulted in tumors with rapid growth consisting of undifferentiated cells. Additionally, riPSCs could be successfully differentiated into a neuronal lineage including glutamate neurons that responded to agonist stimulation. These observations demonstrate a glycophenotypic difference that may potentially serve as a useful probe for riPSC evaluation and to study the role of glycans in pluripotency and carcinogenesis in these cells.

Reprogramming rat embryonic fibroblasts into induced pluripotent stem cells using transposon vectors and their chondrogenic differentiation in vitro.

It has been known that the successful reprogramming of differentiated human somatic cells into a pluripotent state would allow for the creation of cartilage cells. However, current virus‑mediated strategies to form induced pluripotent stem cells (iPSCs) are limited in their clinical application due to exogenous gene modification. In the present study, the piggyBac transposon system carrying corresponding genes (Oct3/4, Klf4, Sox2 and c‑Myc) was employed to reprogram rat embryonic fibroblasts (REFs) into iPSCs, and the transposon‑carried genes were successfully removed by a transposase system. Furthermore, the differentiation capacity of the iPSCs into chondrocytes was investigated in vitro. A typical chondrocyte marker, collagen‑II, was expressed following culture. In conclusion, rat iPSCs without genetic modification were obtained from REFs using the piggyBac transposon system and the transposase mPB system, and these cells possessed the capacity of chondrocyte differentiation, suggesting that this method may be an effective approach for the treatment of cartilage disorders in the future.

Generation of rat-induced pluripotent stem cells from a new model of metabolic syndrome.

We recently characterized DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive rats and Zucker rats, as a new animal model of metabolic syndrome (MetS). Although the phenotype of DS/obese rats is similar to that of humans with MetS, the pathophysiological and metabolic characteristics in each cell type remain to be clarified. Hence, the establishment of induced pluripotent stem cells (iPSCs) derived from MetS rats is essential for investigations of MetS in vitro. Reports of rat iPSCs (riPSCs), however, are few because of the difficulty of comparing to other rodents such as mouse. Recently, the advantage of using mesenchymal stromal cells (MSCs) as a cell source for generating iPSCs was described. We aimed to establish riPSCs from MSCs in adipose tissues of both DS/obese rats and their lean littermates, DahlS.Z-Lepr+/Lepr+ (DS/lean) rats using lentivirus vectors with only three factors Oct4, Klf4, and Sox2 without c-Myc. The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas). Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone. Real-time PCR analysis also revealed that both riPSCs and the adipose tissue from DS/obese and DS/lean rats possess similar expression patterns of adipocyte differentiation-related genes. We succeeded in generating riPSCs effectively from MSCs of both DS/obese and DS/lean rats. These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

Therapeutic effect of TSG-6 engineered iPSC-derived MSCs on experimental periodontitis in rats: a pilot study.

BackgroundWe derived mesenchymal stem cells (MSCs) from rat induced pluripotent stem cells (iPSCs) and transduced them with tumor necrosis factor alpha-stimulated gene-6 (TSG-6), to test whether TSG-6 overexpression would boost the therapeutic effects of iPSC-derived MSCs in experimental periodontitis.MethodsA total of 30 female Sprague-Dawley (SD) rats were randomly divided into four groups: healthy control group (Group-N, n = 5), untreated periodontitis group (Group-P, n = 5), iPS-MSCs-treated and iPSC-MSCs/TSG-6-treated periodontitis groups (Group-P1 and P2, n = 10 per group). Experimental periodontitis was established by ligature and infection with Porphyromonas gingivalis around the maxillae first molar bilaterally. MSC-like cells were generated from rat iPSCs, and transducted with TSG-6. iPSC-MSCs or iPSC-MSCs/TSG-6 were administrated to rats in Group-P1 or P2 intravenously and topically, once a week for three weeks. Blood samples were obtained one week post-injection for the analysis of serum pro-inflammatory cytokines. All animals were killed 3 months post-treatment; maxillae were then dissected for histological analysis, tartrate-resistant acid phosphatase (TRAP) staining, and morphological analysis of alveolar bone loss.ResultsAdministration of iPSC-MSC/TSG-6 significantly decreased serum levels of IL-1β and TNF-α in the Group-P2 rats (65.78 pg/ml and 0.56 pg/ml) compared with those in Group-P (168.31 pg/ml and 1.15 pg/ml respectively) (p<0.05). Both alveolar bone loss and the number of TRAP-positive osteoclasts showed a significant decrease in rats that received iPSC-MSC/TSG-6 treatment compared to untreated rats in Group-P (p<0.05),ConclusionsWe demonstrated that overexpression of TSG-6 in rat iPSC-derived MSCs were capable of decreasing inflammation in experimental periodontitis and inhibiting alveolar bone resorption. This may potentially serve as an alternative stem-cell-based approach in the treatment and regeneration of periodontal tissues.

A Comprehensive System for Generation and Evaluation of Induced Pluripotent Stem Cells Using piggyBac Transposition

The most stringent criterion for evaluating pluripotency is generation of chimeric animals with germline transmission ability. Because the quality of induced pluripotent stem cell (iPSC) lines is heterogeneous, an easy and accurate system to evaluate these abilities would be useful. In this study, we describe a simple but comprehensive system for generating and evaluating iPSCs by single transfection of multiple piggyBac (PB) plasmid vectors encoding Tet-inducible polycistronic reprogramming factors, a pluripotent-cell–specific reporter, a constitutively active reporter, and a sperm-specific reporter. Using this system, we reprogrammed 129 and NOD mouse embryonic fibroblasts into iPSCs, and then evaluated the molecular and functional properties of the resultant iPSCs by quantitative RT-PCR analysis and chimera formation assays. The iPSCs contributed extensively to chimeras, as indicated by the constitutively active TagRFP reporter, and also differentiated into sperm, as indicated by the late-spermatogenesis–specific Acr (acrosin)-EGFP reporter. Next, we established secondary MEFs from E13.5 chimeric embryos and efficiently generated secondary iPSCs by simple addition of doxycycline. Finally, we applied this system to establishment and evaluation of rat iPSCs and production of rat sperm in mouse–rat interspecific chimeras. By monitoring the fluorescence of Acr-EGFP reporter, we could easily detect seminiferous tubules containing rat iPSC–derived spermatids and sperm. And, we succeeded to obtain viable offspring by intracytoplasmic sperm injection (ICSI) using these haploid male germ cells. We propose that this system will enable robust strategies for induction and evaluation of iPSCs, not only in rodents but also in other mammals. Such strategies will be especially valuable in non-rodent species, in which verification of germline transmission by mating is inefficient and time-consuming.