Temporal regulation of Lin28a during mammalian neurulation contributes to neonatal body size control.

BACKGROUND: The timing of developmental events is tightly regulated along a time axis for normal development. Although the RNA-binding protein Lin28a plays a crucial role in the regulation of developmental timing in Caenorhabditis elegans, how the timing of Lin28a expression affects the rate and/or duration of developmental events during mammalian development remains to be addressed. RESULTS: In this study, we discovered that the timing and the duration of Lin28a expression affect embryonic growth. During the neurulation stage of mouse development, endogenous Lin28a levels start to drop. When Lin28a expression was maintained transiently using the inducible tetracycline-regulated gene expression (Tet-ON) system [doxycycline (Dox)-inducible Lin28a transgenic (iLin28a Tg) mice] with Dox administration at E8.5 and E9.5, it resulted in neonatal lethality, increased body weight (organomegaly), and an increased number of caudal vertebrae at birth. On the other hand, Lin28a induction only at E8.5 caused neonatal lethality and organomegaly, but did not affect the caudal vertebra number. Of note, although Dox treatment before or after neurulation still caused neonatal lethality, it neither caused organomegaly nor the increased caudal vertebra number in iLin28a Tg neonates. CONCLUSIONS: Temporal regulation of Lin28a expression during neurulation affects developmental events such as cessation of axial elongation and embryonic growth in mice.

Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy.

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response. Similarly, in vivo cell implantation analysis with siRNA-mediated down-regulation of HLA-E demonstrates that HLA-E is involved in immunosuppression. As hPAE cells efficiently transdifferentiate into myoblasts/myocytes in vitro, we transplanted the cells into mdx mice, a model of Duchenne muscular dystrophy. hPAE cells conferred dystrophin to myocytes of the 'immunocompetent' mdx mice with extremely high efficiency. These findings suggest that HLA-E-expressing cells with a myogenic potential represent a promising source for cell-based therapy of patients with muscular dystrophy.

Xenografted human amniotic membrane-derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes.

RATIONALE: Amniotic membrane is known to have the ability to transdifferentiate into multiple organs and is expected to stimulate a reduced immunologic reaction. OBJECTIVE: Determine whether human amniotic membrane-derived mesenchymal cells (hAMCs) can be an ideal allograftable stem cell source for cardiac regenerative medicine. METHODS AND RESULTS: We established hAMCs. After cardiomyogenic induction in vitro, hAMCs beat spontaneously, and the calculated cardiomyogenic transdifferentiation efficiency was 33%. Transplantation of hAMCs 2 weeks after myocardial infarction improved impaired left ventricular fractional shortening measured by echocardiogram (34+/-2% [n=8] to 39+/-2% [n=11]; P<0.05) and decreased myocardial fibrosis area (18+/-1% [n=9] to 13+/-1% [n=10]; P<0.05), significantly. Furthermore hAMCs transplanted into the infarcted myocardium of Wistar rats were transdifferentiated into cardiomyocytes in situ and survived for more than 4 weeks after the transplantation without using any immunosuppressant. Immunologic tolerance was caused by the hAMC-derived HLA-G expression, lack of MHC expression of hAMCs, and activation of FOXP3-positive regulatory T cells. Administration of IL-10 or progesterone, which is known to play an important role in feto-maternal tolerance during pregnancy, markedly increased HLA-G expression in hAMCs in vitro and, surprisingly, also increased cardiomyogenic transdifferentiation efficiency in vitro and in vivo. CONCLUSIONS: Because hAMCs have a high ability to transdifferentiate into cardiomyocytes and to acquire immunologic tolerance in vivo, they can be a promising cellular source for allograftable stem cells for cardiac regenerative medicine.

Function of EWS-POU5F1 in sarcomagenesis and tumor cell maintenance.

POU5F1 is a transcription factor essential for the self-renewal activity and pluripotency of embryonic stem cells and germ cells. We have previously reported that POU5F1 is fused to EWSR1 in a case of undifferentiated sarcoma with chromosomal translocation t(6;22)(p21;q12). In addition, the EWS-POU5F1 chimeras have been recently identified in human neoplasms of the skin and salivary glands. To clarify the roles of the EWS-POU5F1 chimera in tumorigenesis and tumor cell maintenance, we used small-interfering RNA-mediated gene silencing. Knockdown of EWS-POU5F1 in the t(6;22) sarcoma-derived GBS6 cell line resulted in a significant decrease of cell proliferation because of G1 cell cycle arrest associated with p27(Kip1) up-regulation. Moreover, senescence-like morphological changes accompanied by actin polymerization were observed. In contrast, EWS-POU5F1 down-regulation markedly increased the cell migration and invasion as well as activation of metalloproteinase 2 and metalloproteinase 14. The results indicate that the proliferative activity of cancer cells and cell motility are discrete processes in multistep carcinogenesis. These findings reveal the functional role of the sarcoma-related chimeric protein as well as POU5F1 in the development and progression of human neoplasms.

Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles.

Low efficiencies of nonviral gene vectors, such as transfection reagent, limit their utility in gene therapy. To overcome this disadvantage, we report on the preparation and properties of magnetic nanoparticles [diameter (d) = 121.32 ± 27.36 nm] positively charged by cationic polymer deacylated polyethylenimine (PEI max), which boosts gene delivery efficiency compare with polyethylenimine (PEI), and their use for the forced expression of plasmid delivery by application of a magnetic field. Magnetic nanoparticles were coated with PEI max, which enabled their electrostatic interaction with negatively charged molecules such as plasmid. We successfully transfected 81.1 ± 4.0% of the cells using PEI max-coated magnetic nanoparticles (PEI max-nanoparticles). Along with their superior properties as a DNA delivery vehicle, PEI max-nanoparticles offer to deliver various DNA formulations in addition to traditional methods. Furthermore, efficiency of the gene transfer was not inhibited in the presence of serum in the cells. PEI max-nanoparticles may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.

Cells of extraembryonic mesodermal origin confer human dystrophin in the mdx model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is an X-linked recessive genetic disease characterized by severe skeletal muscular degeneration. The placenta is considered to be a promising candidate cell source for cellular therapeutics because it contains a large number of cells and heterogenous cell populations with myogenic potentials. We analyzed the myogenic potential of cells obtained from six parts of the placenta, that is, umbilical cord, amniotic epithelium, amniotic mesoderm, chorionic plate, villous chorion, and decidua basalis. In vitro cells derived from amniotic mesoderm, chorionic plate, and villous chorion efficiently transdifferentiate into myotubes. In addition, in vivo implantation of placenta-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of human dystrophin. Differential contribution to myogenesis in this study may be attributed to placental portion-dependent default cell state. Molecular taxonomic characterization of placenta-derived maternal and fetal cells in vitro will help determine the feasibility of cell-based therapy.

Efficient reprogramming of human and mouse primary extra-embryonic cells to pluripotent stem cells.

Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS.

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.

Rapid detection of ketamine and norketamine in rat hair using micropulverized extraction and ultra-performance liquid chromatography-electrospray ionization mass spectrometry.

A new method for the rapid and simultaneous detection of ketamine and its major metabolite, norketamine, in rat hair has been developed by combining micropulverized extraction and ultraperformance liquid chromatography-electrospray ionization mass spectrometry. By using reversed-phase UPLC, ketamine and norketamine were well separated within 2 min. Using ketamine-dosed rat hair, the conditions for micropulverized extraction were optimized, and the limits of detection and quantification of the developed method were found to be 1.7 and 5.7 pg/mg hair for ketamine, respectively. The precisions achieved with this method were slightly better than that obtained with conventional acidic methanol extraction method. Using this proposed method, analysis of the washed rat hair could be completed within 16-17 min. This method is expected to be applied for the analysis of the hair samples of not only rats but also ketamine abusers.

Amnion-derived cells as a reliable resource for next-generation regenerative medicine.

The placenta is composed of the amnion, chorionic plate, villous and smooth chorion, decidua basalis, and umbilical cord. The amnion is a readily obtainable source of a large number of cells and cell types, including epithelium, mesenchyme, and endothelium, and is thus an allogeneic resource for regenerative medicine. Endothelial cells are obtained from large arteries and veins in the amniotic membrane as well as the umbilical cord. The amnion-derived cells exhibit transdifferentiation capabilities, including chondrogenesis and cardiomyogenesis, by introduction of transcription factors, in addition to their original and potential phenotypes. The amnion is also a source for production of induced pluripotent stem cells (AM-iPSCs). The AM-iPSCs exhibit stable phenotypes, such as multipotency and immortality, and a unique gene expression pattern. Through the use of amnion-derived cells, as well as other placenta-derived cells, preclinical proof of concept has been achieved in a mouse model of muscular dystrophy.

Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1.

CCN3/NOV activates the Notch signal through the carboxyl terminal cysteine-rich (CT) domain. CCN3 transfection to Kusa-A1 inhibited osteogenic differentiation and cell proliferation, which is accompanied by upregulation of Hes/Hey, Notch downstream targets, and p21, a CDK inhibitor. Upregulation of Hes/Hey and p21 was abrogated by the deletion of CT domain. Anti-proliferative activity of CCN3 was also abrogated by CT domain deletion whereas anti-osteogenic activity was not completely abrogated. We found that CT domain-deleted CCN3 still possesses antagonistic effect on BMP-2. These results suggest that CCN3 employs Notch and BMP pathways in anti-osteogenic activity while it inhibits cell proliferation uniquely by Notch/p21 pathway.

Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential.

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.

[Cell source for regenerative medicine].

Mesenchymal stem cells (MSCs) are a potential cellular source for stem cell-based therapy, since they have the ability to proliferate and differentiate into mesodermal tissues. Human MSCs have been used clinically to treat patients with graft versus host disease and osteogenesis imperfecta. We previously showed that murine and human marrow-derived MSCs can differentiate into cardiomyocytes, skeletal myocytes, osteoblasts, chondroblasts, adipocytes, and neuron. We here show that sources of MSCs with multipotency includes placenta, endometrium, menstrual blood, umbilical cord, cartilage and so on. Differentiation potentials of MSCs depend on cell source, implying that MSCs obtained from each source have differential default state ex vivo.

Developmental pluripotency of the nuclei of neurons in the cerebral cortex of juvenile mice.

Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14-21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.

Mouse embryos and chimera cloned from neural cells in the postnatal cerebral cortex.

Cloning of mice has been achieved by transferring nuclei of various types of somatic cell nuclei into enucleated oocytes. However, all attempts to produce live cloned offspring using the nuclei of neurons from adult cerebral cortex have failed. Previously we obtained cloned mice using the nuclei of neural cells collected from fetal cerebral cortex. Here, we attempted to generate cloned mice using differentiated neurons from the cerebral cortex of postnatal (day 0-4) mice. Although we were unable to obtain live cloned pups, many fetuses reached day 10.5 days of development. These fetuses showed various abnormalities such as spherical omission of the neuroepithelium, collapsed lumen of neural tube, and aberrant expressions of marker proteins of neurons. We produced chimeric mice in which some hair cells and kidney cells were originated from differentiated neurons. In chimeric fetuses, LacZ-positive donor cells were in all three germ cell layers. However, chimeras with large contribution of donor-derived cells were not obtained. These results indicate that nuclei of differentiated neurons have lost their developmental totipotency. In other words, the conventional nuclear transfer technique does not allow nuclei of differentiated neurons to undergo complete genomic reprogramming required for normal embryonic development.

Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).

Isolation and characterization of the human immature osteoblast culture system from the alveolar bones of aged donors for bone regeneration therapy.

BACKGROUND: Establishment of human osteoblast cultures that retain bone-forming capacity is one of the prerequisites for successful bone regeneration therapy. Because osteoblasts harvested from adults exhibit limited growth, the use of immature osteoblasts that can expand ex vivo should greatly facilitate bone regeneration therapy. In this study, we developed immature human osteoblasts isolated from aged alveolar bone (HAOBs). METHODS: HAOBs obtained after the collagenase digestion of alveolar bones from elderly donors. Then, we assessed osteogenic ability of HAOB after treatment with recombinant human bone morphogenic protein-2 or transplantation into immunodeficient mice. In addition, we performed global gene expression analysis to identify functional marker for HAOB. RESULTS: HAOBs, which can differentiate into osteoblasts and have a robust bone-forming ability, were successfully extracted from donors who were > 60 years of age. We found that the HAOBs exhibited a higher osteogenic ability compared with those of human mesenchymal stem cells and highly expressed NEBULETTE (NEBL) with osteogenic abilities. CONCLUSIONS: HAOBs have properties similar to those of human immature osteoblasts and appear to be a novel material for cell-based bone regeneration therapy. Additionally, the expression level of NEBL may serve as a marker for the osteogenic ability of these cells.

Properties and usefulness of aggregates of synovial mesenchymal stem cells as a source for cartilage regeneration.

INTRODUCTION: Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. METHODS: Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. RESULTS: Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. CONCLUSIONS: Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.

Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors.

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. We hypothesized that combinatorial expression of chondrocyte-specific transcription factors could directly convert human placental cells into chondrocytes. Starting from a pool of candidate genes, we identified a combination of only five genes (5F pool)-BCL6, T (also called BRACHYURY), c-MYC, MITF, and BAF60C (also called SMARCD3)-that rapidly and efficiently convert postnatal human chorion and decidual cells into chondrocytes. The cells generated expressed multiple cartilage-specific genes, such as Collagen type II α1, LINK PROTEIN-1, and AGGRECAN, and exhibited characteristics of cartilage both in vivo and in vitro. Expression of the endogenous genes for T and MITF was initiated, implying that the cell conversion is due to not only the forced expression of the transgenes, but also to cellular reprogramming by the transgenes. This direct conversion system from noncartilage tissue to cartilaginous tissue is a substantial advance toward understanding cartilage development, cell-based therapy, and oncogenesis of chondrocytes.

Establishment of functioning human corneal endothelial cell line with high growth potential.

Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+)- and K(+)-dependent ATPase (Na(+)/K(+)-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+)/K(+)-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.