Positive Alkaline Phosphatase Staining Research Articles

Bacterial Ribosomes Induce Plasticity in Mouse Adult Fibroblasts.

The incorporation of bacterial ribosome has been reported to induce multipotency in somatic and cancer cells which leads to the conversion of cell lineages. Queried on its universality, we observed that bacterial ribosome incorporation into trypsinized mouse adult fibroblast cells (MAF) led to the formation of ribosome-induced cell clusters (RICs) that showed strong positive alkaline phosphatase staining. Under in vitro differentiation conditions, RICs-MAF were differentiated into adipocytes, osteoblasts, and chondrocytes. In addition, RICs-MAF were able to differentiate into neural cells. Furthermore, RICs-MAF expressed early senescence markers without cell death. Strikingly, no noticeable expression of renowned stemness markers like Oct4, Nanog, Sox2, etc. was observed here. Later RNA-sequencing data revealed the expression of rare pluripotency-associated markers, i.e., Dnmt3l, Sox5, Tbx3 and Cdc73 in RICs-MAF and the enrichment of endogenous ribosomal status. These observations suggested that RICs-MAF might have experienced a non-canonical multipotent state during lineage conversion. In sum, we report a unique approach of an exo-ribosome-mediated plastic state of MAF that is amenable to multi-lineage conversion.

Establishment and Characterization of a Spermatogonial Stem Cell Line from Tiger Puffer Fish (Takifugu rubripes).

Tiger puffer fish (Takifugu rubripes) has become the main fish species cultured in China since the last century because of its high economic value. Male and female tiger puffer fish need 2 and 3 years each to reach sexual maturity, which limits the development of breeding research for this species. In recent years, in vitro culture of fish spermatogonial stem cells (SSCs) have shown potential in aquaculture. In the present study, we established a spermatogenic stem cell line from T. rubripes (TrSSCs). TrSSCs were characterized by polygonal morphology, predominantly retained 44 chromosomes, and grew rapidly at 26 °C and in L-15. TrSSCs were still able to grow stably after more than one year of in vitro culture. TrSSCs showed positive alkaline phosphatase staining. TrSSCs expressed germ cell-associated genes, including dnd, ddx4, piwil, gfra1b, sox2, myca, nanog, ly75, and dazl, as determined by semiquantitative assays, and almost all cells were found to express the germ cell genes ddx4 and gfra1b in a fluorescence in situ hybridization assay. In vitro, induction experiments demonstrated the TrSSCs possessed the ability to differentiate into other types of cells. Our research has enriched the fish spermatogonial stem cell resource bank, which will provide an efficient research model for sex determination and sex control breeding in fish, establishing a foundation for subsequent breeding research.

Identification of potential regulatory genes for embryonic stem cell self-renewal and pluripotency by random forest

To identify novel genes associated with self-renewal and pluripotency of mouse embryonic stem cells(mESCs)by integrating multiomics data based on machine learning methods. We integrated multiomics information of mESCs involving transcriptome, histone modifications, chromatin accessibility, transcription factor binding and architectural protein binding, and compared the signal differences between known stem cell self-renewal and pluripotency genes and other genes.By integrating these multiomics data, we established prediction models based on several machine learning classifiers including random forests and performed 5-fold cross validations.The model was trained using the training dataset containing two thirds of the input samples, and the remaining one third of the input samples were used as the test dataset to assess the performance of the model in independent tests.Finally, the results predicted by the model were validated through gene function annotation and cell function experiments including cell viability assay, colony formation assay and cell cycle analysis. Compared with the random genes, the genes known to be associated with self-renewal and pluripotency of mESCs in the multiomics data showed significantly different features.Random forest outperformed the other machine learning algorithms tested on these multiomics data, with an area under the curve (AUC) of 0.883±0.018 for cross validation and an AUC of 0.880±0.028 for independent test.Based on this model, we identified 893 potential regulatory genes associated wwith self-renewal and pluripotency of mESCs, which were similar to the known genes in functional annotation.Known-down of the predicted novel regulator gene Cct6a resulted in significant decreases in the cell viability of mESCs (P < 0.0001) and the number of cell clones (P < 0.01), significantly increased the number of cells in G1 phase (P < 0.01) and decreasedthe number of S phase cells (P < 0.05).Knockdown of Cct6a also led to failure of positive alkaline phosphatase staining of the mESCs. Machine learning model based on multiomics data can be used to predict potential self-renewal and pluripotency regulators with high performance.By using this model, we predicted potential self-renewal and pluripotency regulatory genes including Cct6a and applied experimental validation.This model provides new insights into the regulatory mechanism of mESCs and contribute to stem cell research.

The Subacromial Bursa is a Viable Source of Autologous Mesenchymal Stem Cells for Rotator Cuff Repair

Objectives: Chronic rotator cuff tears still represent a significant source of morbidity and functional decline in the general population. The purpose of this study was to establish protocols for isolation and expansion of bursa-derived mesenchymal stem cells (BDSCs) and to evaluate their differentiation capacity, including tenogenesis. We hypothesized that BDSCs would be capable of multilineage differentiation (including tenogenesis) and represent an important source for autologous stem cells for patients undergoing rotator cuff repair. Methods: After IRB approval, 10 patients (ages 43-65 years) scheduled to undergo arthroscopic repair for chronic rotator cuff tears were enrolled. During diagnostic arthroscopy, subacromial bursa tissue was harvested using an arthroscopic shaver and collected by attaching the outflow tubing to a specialized specimen cup. Tissue specimens were transported to our laboratory for analysis. BDSCs were isolated via adherent culture and plated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Chondrogenic, adipogenic, and osteogenic induction media were used to induce differentiation. Tenogenic induction was performed using DMEM supplemented with varying concentrations of BMP-12, ascorbic acid, and human tenocyte-conditioned media. Alcian Blue staining was used to evaluate chondrogenesis, Oil Red O staining for adipogenesis, and Alkaline Phosphatase staining for osteogenesis. Gene expression markers for adipogenesis (ADIPOQ, FABP4, PPARγ), chondrogenesis (COL2A1 and SOX5), and osteogenesis (osteocalcin, osterix), along with primary antibodies to tenogenic markers (scleraxis, tenomodulin), were used to verify each cell lineage. Results: BDSCs isolated by adherent culture without collagen exhibited a spindle-shaped morphology characteristic of mesenchymal stem cells (MSCs), formed colonies, and demonstrated great expandability for six to eight passages without morphology changes (Figure 1A). After 3 weeks of culture, 95% (p&lt;0.0001) of the BDSCs expressed the MSC surface marker CD90 and were negative for non-MSC cell markers (CD45, CD146, CD31, and CD34) (Figure 1B and C). These BDSCs demonstrated a capacity for adipogenesis (positive Oil Red O staining, ADIPOQ, FABP4, PPARγ expression), osteogenesis (positive alkaline phosphatase staining, positive osteocalcin and scleraxis expression), chondrogenesis (positive Alcian Blue staining, positive COL2A1 and SOX5 expression), and tenogenesis (scleraxis and tenomodulin expression) (Figure 2). The results indicate that BDSCs are multipotent as evidenced by their differentiation into fat, bone, cartilage, and tendon cells. For tenogenesis, we found that 7-day incubation in DMEM supplemented with 100 ng/mL of BMP-12 and 50 μg/mL of ascorbic acid produced superior tenogenic induction. These BDSCs adapted an elongated morphology combined with the expression of both scleraxis and tenomodulin - a unique characteristic of native tenocytes. Conclusion: Our results demonstrate that subacromial bursa represents a viable source of mesenchymal stem cells. We developed a reliable protocol for isolation of BDSCs from patient bursa samples. We show that BDSCs in the presence of BMP-12 and ascorbic acid can differentiate toward a tenogenic lineage. Our work provides strong evidence that BDSCs may be a potent tool for cellular therapy and may benefit future patients who undergo surgical repair of chronic rotator cuff tears. [Figure: see text][Figure: see text]

Idiopathic Inflammatory Myopathy and Anti-aminoacyl-tRNA Synthetase Antibody

Anti-aminoacyl-tRNA synthetase (ARS) antibodies (anti-Jo-1, anti-PL-7, anti-PL-12, anti-EJ, anti-KS, anti-OJ, anti-Ha, and anti-Zo antibodies) are found in 25 to 40% of myositis patients. The patients with these antibodies have anti-synthetase syndrome with one or more of the following clinical features: myositis, interstitial lung disease, arthritis, fever, Raynaud's phenomenon, and mechanic's hands. In Japan, health insurance coverage of treatments for patients in whom the "anti-ARS antibodies," anti-Jo-1, anti-PL-7, anti-PL-12, anti-EJ, and anti-KS are detected by enzyme-linked immunosorbent assay was approved by the Ministry of Health, Labour and Welfare in 2014. Recently, clinical features have been discovered to be heterogeneous. Patients with the anti-PL-7, anti-PL-12, anti-KS, and anti-OJ antibodies exhibit interstitial lung disease rather than myositis. Interstitial lung disease is related to the prognosis of this syndrome. Regarding histopathological findings of the muscle, perimysial connective tissue fragmentation with positive staining for alkaline phosphatase is the characteristic feature. Myonuclear actin filament inclusions are also detected. A recent work demonstrated that immunization of mice with histidyl-tRNA synthetase results in muscle inflammation consistent with myositis. These findings promote understanding of the pathological mechanism of the development of myositis associated with anti-ARS antibodies.

Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes

To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes. We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method. Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively. We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

3D Culture of rat embryonic stem cells and rat induced pluripotent stem cells in a novel PGmatrix hydrogel (LB20)

Expanding embryonic stem cells (ESCs) in a synthetic 3D microenvironment in vitro while maintaining the undifferentiated state has been challenging. It would be advantageous to identify a substrate which could be modified such as by changing ligands that interact with cell surface receptors or by changing the substrate’s stiffness. We developed a synthetic PGmatrix hydrogel to include these design specifications and determined whether it could support the expansion of undifferentiated ESCs in a 3D microenvironment. Rat ESCs (rESCs) and rat induced pluripotent stem cells (riPSCs) were used for evaluation. The cells were grown both in 2D and 3D in PGmatrix hydrogel containing 2 inhibitors. Dynamic rheological tests of PGmatrix hydrogel in 2i medium showed a rapid increase of storage moduli (G’ = 600 Pa for 3mM in 1 hr) indicating gelation after mixing. The mechanical strength of hydrogel was full recovered after shear‐thinning into liquid phase.Also, the hydrogel reached a self‐supporting strength within 1000sec which is a reasonable rate to ensure cells were suspended in the hydrogel matrix before they reached the bottom of the plate. Both cell lines were cultured for more than 30 passages using regular trypsinization method to produce single cells at passage prior to plating. We harvested cells from the hydrogel for passaging by shearing the gel to liberate the cells. Phase contrast images of cells grown in 3D showed similar morphology which had rounded phase‐bright borders compared to cells grown on murine embryonic fibroblast (MEF) feeder layer. They retained pluripotent stem cell properties and showed positive staining for alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. Karyotypes of pluripotent stem cells cultured in 3D were normal after 20 passages. Embryoid bodies (EBs) could be formed by hanging drop after the cells were trypsinized from 3D cultures. We plan to compare the gene expression profiles of 2D and 3D cells and determine if the 3D cultured cells can undergo differentiation into all the 3 germ layers and form teratomas. Growing ESCs in a fully synthetic 3D culture can significant advantages for directing differentiation and examining the interactions with surface receptors. Such knowledge will enable 3D culture for their use in drug development or for encapsulation of cells for clinical use.

Generation of chimeric piglets by injection of embryonic germ cells from inbred Wuzhishan miniature pigs into blastocysts

Human embryonic stem/germ (ES/EG) cell research poses ethical dilemma, it is therefore critical to establish alternative sources of cells for relevant studies. Considering the similarities between the inbred miniature Wuzhishan pigs (WZSP) and humans, ES/EG from these pigs can serve as potential substitutes in human research. In this study, we reported our results that successfully established stable EG cell lines from the WZSP. Primordial germ cells (PGCs) were isolated from the genital ridges of pig fetuses at 25 to 28days of pregnancy. To obtain stable EG cell line, PGCs were maintained on STO cells in DMEM containing multiple essential growth factors. Two EG cell lines were established and characterized by positive alkaline phosphatase staining (AKP), expressions of Oct-4, SSEA-1, SSEA-3, SSEA-4, ability to differentiate into cells of all three germ layers in vitro, and generation of chimeric offsprings after microinjection and embryo transfer. Transmission electron microscopy demonstrated that the cytoplasmic structure of pig EG cells were rather simple and had a higher nuclear-to-cytoplasm ratio. Scanning electron microscopy showed the sizes of pig EG cells were similar to mouse EG cells. Both EG cell lines showed normal karyotypes. The EG cells were propagated for more than 20 passages and underwent multiple cycles of freezing and thawing, without losing their pluripotency (as distinguished by AKP staining). Both in vitro and in vivo evidence strongly demonstrated that EG cells harvested from the inbred miniature WZSP were pluripotent and can be used for relevant pig or human studies.

Fabrication and in vitro evaluation of a sponge-like bioactive-glass/gelatin composite scaffold for bone tissue engineering

In this work a bioactive composite scaffold, comprised of bioactive-glass and gelatin, is introduced. Through direct foaming a sponge-like composite of a sol–gel derived bioactive-glass (70S30C; 70% SiO2, 30% CaO) and porcine gelatin was developed for use as a biodegradable scaffold for bone tissue engineering. The composite was developed to provide a suitable alternative to synthetic polymer based scaffolds, allowing directed regeneration of bone tissue. The fabricated scaffold was characterised through X-ray microtomography, scanning electron and light microscopy demonstrating a three dimensionally porous and interconnected structure, with an average pore size (170μm) suitable for successful cell proliferation and tissue ingrowth. Acellular bioactivity was assessed through apatite formation during submersion in simulated body fluid (SBF) whereby the rate and onset of apatite nucleation was found to be comparable to that of bioactive-glass. Modification of dehydrothermal treatment parameters induced varying degrees of crosslinking, allowing the degradation of the composite to be tailored to suit specific applications and establishing its potential for a wide range of applications. Use of genipin to supplement crosslinking by dehydrothermal treatment provided further means of modifying degradability. Biocompatibility of the composite was qualified through successful cultures of human dental pulp stem cells (HDPSCs) on samples of the composite scaffold. Osteogenic differentiation of HDPSCs and extracellular matrix deposition were confirmed through positive alkaline phosphatase staining and immunohistochemistry.

The Effect of rhBMP-4 on Alkaline Phosphatase Expression of Gingival Fibroblast

This study examined whether the alkaline phosphatase of human gingival fibroblast is enhanced by recombinant human BMP-4. Gingival fibroblast was obtained from the excised gingival tissue of an implant patient undergoing gingival surgery. The tissue was incubated at 37° in 5% CO2 and 95% humidity, and the cultivating media was changed every 2 days. The 2nd passage gingival fibroblasts were cultured in a medium containing Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1 X antibiotic antimycotic solution. The control gingival fibroblast was cultured without rhBMP-4. The experimental group 1 were cultured with low-dose rhBMP-4. The experimental group 2 were cultured with high-dose rhBMP-4. This study evaluated the effect of rhBMP-4 on alkaline phosphatase expression of gingival fibroblast using alkaline phosphatase assay. In the experimental group 2 with low dose rhBMP-4, the gingival fibroblast showed abundant positive alkaline phosphatase staining. In the control group, the gingival fibroblast showed weak positive alkaline phosphatase staining. Overall, these results suggest that the alkaline phosphatase expression of human gingival fibroblast can enhanced by low dose rhBMP-4.

Mouse Induced Pluripotent Stem Cells Generated by piggyBac

Mouse somatic cells have been successfully reprogrammed into embryonic stem cell-like cells by enforced expression of defined transcription factors, and the reprogrammed cells are termed as induced pluripotent stem cells. Such cell reprogramming strategy is involved in gene transfection with retroviral, lentiviral, adenoviral and plasmid vectors. In addition, proteins and small molecules, synthetic modified mRNA and microRNA also have been used to efficiently generate mouse and human iPS. As a high efficient transposon, piggyBac has been used to generate induced pluripotent stem cells. In this study, piggyBac carrying four transcription factors called Oct4 , Sox2 , Klf4 and c-Myc has been employed to generate mouse induced pluripotent stem cells from murine embryonic fibroblasts, and the differentiation potential of the cells was assessed in vivo and in vitro . The reprogrammed cells showed positive staining for alkaline phosphatase and positive for OCT4, SSEA-1 and NANOG by immunofluorescent analyses. RT-PCR results showed that Oct4 , Sox2 , Klf4 and c-Myc was expressed in the reprogrammed cells. Immunofluorescent staining of AFP, BRACHYURY and NCAM1 indicated that the cells have the potential to differentiate spontaneously into all three germ layers in vitro . Teratomas formed after the cells subcutaneously injected into NOD-SCID mice. Chimeric blastocysts were developed when the cells were aggregated with mouse 8-cell embryos. These results suggested that mouse induced pluripotent stem cells can be generated from fibroblast cells by piggyBac transfection system. Such induced pluripotent stem cells are similar to mouse ES cells and have potential to differentiate into three germ layers in vitro and in vivo .

Mitochondrial fusion by pharmacological manipulation impedes somatic cell reprogramming to pluripotency: New insight into the role of mitophagy in cell stemness

Recent studies have suggested a pivotal role for autophagy in stem cell maintenance and differentiation. Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has been also suggested to bio-energetically take advantage of mitochondrial autophagy (mitophagy). We have preliminary addressed how mitophagy might play a role in the regulation of induced pluripotency using mdivi-1 (for mitochondrial division inhibitor), a highly efficacious small molecule that selectively inhibits the self-assembly of DRP1, a member of the dynamin family of large GTPases that mediates mitochondrial fission. At mdivi-1 concentrations that rapidly induced the formation of mitochondrial net-like or collapsed perinuclear mitochondrial structures, we observed that the reprogramming efficiency of mouse embryonic fibroblasts transduced with the Yamanaka three-factor cocktail (OCT4, KLF4, and SOX2) is drastically reduced by more than 95%. Treatment of MEFs with mdivi-1 at the early stages of reprogramming before the appearance of iPSC colonies was sufficient to completely inhibit somatic cell reprogramming. Therefore, the observed effects on reprogramming efficiencies were due likely to the inhibition of the process of reprogramming itself and not to an impairment of iPSC colony survival or growth. Moreover, the typical morphology of established iPSC colonies with positive alkaline phosphatase staining was negatively affected by mdivi-1 exposure. In the presence of mdivi-1, the colony morphology of the iPSCs was lost, and they somewhat resembled fibroblasts. The alkaline phosphatase staining was also significantly reduced, a finding that is indicative of differentiation. Our current findings provide new insight into how mitochondrial division is integrated into the reprogramming factors-driven transcriptional network that specifies the unique pluripotency of stem cells.

Immortalization and characterization of osteoblast cell lines generated from wild‐type and Nmp4‐null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT)

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism.

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.

Activation of pluripotency-associated genes in mouse embryonic fibroblasts by non-viral transfection with in vitro-derived mRNAs encoding Oct4, Sox2, Klf4 and cMyc

The first successful reprogramming of differentiated cells to a pluripotent state was done by retroviral introduction of four transcription factors (Oct4, Sox2, Klf4, cMyc) by the group of Yamanaka in 2006. Since then, scientists all over the world have attempted various methods to avoid insertional mutagenesis, a major limitation of the retrovirus-based method, however no technique was found to completely avoid DNA integration. Recently, a non-viral mRNA-based approach, inherent to avoid genomic integration, was implemented to generate stem cell-like cells, yet, seventeen daily transfections were required, inducing substantial stress on the cells. In this work, we demonstrate successful activation of pluripotency-associated genes in mouse embryonic fibroblasts by means of cationic lipid-mediated introduction of mRNAs encoding the four factors. Moreover, our transfection protocol required maximally three transfections. Up-regulation of the transfected factors as well as Nanog and SSEA-1, typical mouse pluripotency markers, was detected already after the first transfection. Nuclear localization of the introduced factors was confirmed. Positive alkaline phosphatase staining of cell clusters further confirmed the onset of the reprogramming process. In conclusion, the transfection method presented here holds great promise for safe generation of induced pluripotent stem cells of mouse origin.

Efficient differentiation of newly derived human embryonic stem cells from discarded blastocysts into hepatocyte‐like cells

To establish an optimal method to isolate and culture human embryonic stem (hES) cells from discarded blastocysts and to differentiate these cells into hepatocyte-like cells. Discarded human blastocysts of days 5-6 were cultured on mouse embryonic fibroblast cell feeder layers. Cells from the inner cell mass were isolated and subsequently cultured in vitro. To induce the formation of embryoid bodies (EB), dissociated ES cells were cultured in hanging drops for 5 days. The resulting EB were plated onto 100 mm plastic gelatin-coated dishes and allowed to attach for the outgrowth culture in the presence of hepatocyte growth factor for an additional 15 days. The hES cells had typical morphological characteristics: round or elliptic nest-like colonies with distinct cell borders and a smooth surface, significant swelling growth, compact cell aggregation, and relatively big nucleus compared to a small cytoplasm. The hES cells were identified by positive alkaline phosphatase staining. All had normal karyotypes and expressed octamer-binding transcription factor and cell surface markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, and tumor rejection antigens TRA-1-60 and TRA-1-81. The cells could be differentiated to form teratomas in vivo. The hES cells could be induced to differentiate into hepatocyte-like cells and 70-80% of the cells expressed liver-associated proteins. Three hES cell lines have been successfully isolated and cultured from discarded human blastocysts. The hES cells can be efficiently differentiated into hepatocyte-like cells using the EB system. These cells possess the potential for treatment of liver diseases.

Generation of Human Induced Pluripotent Stem Cells from Umbilical Cord Matrix and Amniotic Membrane Mesenchymal Cells

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.

Comparison of Multipotent Differentiation Potentials of Murine Primary Bone Marrow Stromal Cells and Mesenchymal Stem Cell Line C3H10T1/2

Murine C3H10T1/2 cells have many features of mesenchymal stem cells (MSCs). Whether or not the multipotent differentiation capability of C3H10T1/2 cells is comparable to that of primary bone marrow-derived MSCs (BM-MSCs) was investigated in this study. For in vitro osteogenic differentiation, both BM-MSCs and C3H10T1/2 cells differentiated to osteoblastic cell lineage and showed positive staining for alkaline phosphatase (ALP) and increased mRNA expression of Runx2, Col1alphaI, and osteocalcin. C3H10T1/2 cells and BM-MSCs induced similar amounts of bone formation in the biomaterials. Under chondrogenic induction in the presence of TGF-beta1, cell pellets of both BM-MSCs and C3H10T1/2 cells formed cartilage-like tissues with cartilage matrix components including proteoglycan, type II collagen, and aggrecan. However, C3H10T1/2 cells presented lower adipogenic differentiation potential, with only about 10% C3H10T1/2 cells (but about 70% of BM-MSCs) being committed to adipogenesis. In this study we confirmed that C3H10T1/2 cells coimplanted with osteoconductive scaffolds can form bone spontaneously in vivo and that C3H10T1/2 cells have a basal level of osteocalcin expression, suggesting that they may be a good alternative source of primary BM-MSCs for investigating osteogenic and chondrogenic differentiation in bone or cartilage tissue engineering studies. Caution is needed when using C3H10T1/2 cells for adipogenic studies as they appear to have lower adipogenic potential than BM-MSCs.

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. The lack of rat embryonic stem (ES) cells has restricted the availability of transgenic technologies to create genetic models in this species. Here, we show that rat ES cells can be efficiently derived, propagated, and genetically manipulated in the presence of small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. These rat ES cells express pluripotency markers and retain the capacity to differentiate into derivatives of all three germ layers. Most importantly, they can produce high rates of chimerism when reintroduced into early stage embryos and can transmit through the germline. Establishment of authentic rat ES cells will make possible sophisticated genetic manipulation to create models for the study of human diseases.

Cementum–periodontal ligament complex regeneration using the cell sheet technique

In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament-cementum complex. Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature-responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral-like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum-like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum-like tissue. This study suggests that the multilayered temperature-responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament-cementum complex.