Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors.

Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Implication of HpEts in gene regulatory networks responsible for specification of sea urchin skeletogenic primary mesenchyme cells.

The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.

Osteogenic properties of human myogenic progenitor cells.

Here, we identified human myogenic progenitor cells coexpressing Pax7, a marker of muscle satellite cells and bone-specific alkaline phosphatase, a marker of osteoblasts, in regenerating muscle. To determine whether human myogenic progenitor cells are able to act as osteoprogenitor cells, we cultured both primary and immortalized progenitor cells derived from the healthy muscle of a nondystrophic woman. The undifferentiated myogenic progenitors spontaneously expressed two osteoblast-specific proteins, bone-specific alkaline phosphatase and Runx2, and were able to undergo terminal osteogenic differentiation without exposure to an exogenous inductive agent such as bone morphogenetic proteins. They also expressed the muscle lineage-specific proteins Pax7 and MyoD, and lost their osteogenic characteristics in association with terminal muscle differentiation. Both myoblastic and osteoblastic properties are thus simultaneously expressed in the human myogenic cell lineage prior to commitment to muscle differentiation. In addition, C3 transferase, a specific inhibitor of Rho GTPase, blocked myogenic but not osteogenic differentiation of human myogenic progenitor cells. These data suggest that human myogenic progenitor cells retain the capacity to act as osteoprogenitor cells that form ectopic bone spontaneously, and that Rho signaling is involved in a critical switch between myogenesis and osteogenesis in the human myogenic cell lineage.

Expression of an acyl-CoA synthetase, lipidosin, in astrocytes of the murine brain and its up-regulation during remyelination following cuprizone-induced demyelination.

Lipidosin is an 80-kDa protein with long-chain acyl-CoA synthetase activity expressed in the brain, adrenal gland, testis, and ovary, which are selectively damaged in X-linked adrenoleukodystrophy (X-ALD). Western blot analysis of the cerebrum and cerebellum revealed a gradual increase in the expression of lipidosin postnatally. Light microscopic immunohistochemistry using a panel of specific monoclonal antibodies showed that the lipidosin-immunopositive cells were ubiquitously distributed in the brain and were denser in the gray matter than in the white matter. Lipidosin immunoreactivity was colocalized with GFAP immunoreactivity but not with ubiquitin C-terminal hydrolase 1 (= PGP9.5) immunoreactivity, a neuronal marker, and lipidosin-producing cells detected by an antisense probe specific for lipidosin mRNA were also GFAP immunopositive. These data together with Western blot analysis of primary cultured astrocytes indicate that lipidosin is expressed in astrocytes. Immunoelectron microscopic analysis revealed that lipidosin immunoreactivity was widely distributed from perivascular endfeet to perisynaptic processes without being limited to peroxisomes. Lipidosin immunoreactivity was greatly increased in astrocytes in the area of remyelination following experimental demyelination induced by the administration of cuprizone to mice. These data suggest that lipidosin was involved in fatty acid metabolism during reconstruction of the myelin sheath.