Ryoko Araki

Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Equivalency of Nuclear Transfer‐Derived Embryonic Stem Cells to Those Derived from Fertilized Mouse Blastocysts

Therapeutic cloning, whereby nuclear transfer (NT) is used to generate embryonic stem cells (ESCs) from blastocysts, has been demonstrated successfully in mice and cattle. However, if NT‐ESCs have abnormalities, such as those associated with the offspring produced by reproductive cloning, their scientific and medical utilities might prove limited. To evaluate the characteristics of NT‐ESCs, we established more than 150 NT‐ESC lines from adult somatic cells of several mouse strains. Here, we show that these NT‐ESCs were able to differentiate into all functional embryonic tissues in vivo. Moreover, they were identical to blastocyst‐derived ESCs in terms of their expression of pluripotency markers in the presence of tissue‐dependent differentially DNA methylated regions, in DNA microarray profiles, and in high‐coverage gene expression profiling. Importantly, the NT procedure did not cause irreversible damage to the nuclei. These similarities of NT‐ESCs and ESCs indicate that murine therapeutic cloning by somatic cell NT can provide a reliable model for preclinical stem cell research.

DNA-dependent Protein Kinase-independent Activation of p53 in Response to DNA Damage

Phosphorylation at serine 15 of the human p53 tumor suppressor protein is induced by DNA damage and correlates with accumulation of p53 and its activation as a transcription factor. The DNA-dependent protein kinase (DNA-PK) can phosphorylate serine 15 of human p53 and the homologous serine 18 of murine p53in vitro. Contradictory reports exist about the requirement for DNA-PK in vivo for p53 activation and cell cycle arrest in response to ionizing radiation. While primary SCID (severe combined immunodeficiency) cells, that have defective DNA-PK, show normal p53 activation and cell cycle arrest, a transcriptionally inert form of p53 is induced in the SCID cell line SCGR11. In order to unambiguously define the role of the DNA-PK catalytic subunit (DNA-PKcs) in p53 activation, we examined p53 phosphorylation in mouse embryonic fibroblasts (MEFs) from DNA-PKcs-null mice. We found a similar pattern of serine 18 phosphorylation and accumulation of p53 in response to irradiation in both control and DNA-PKcs-null MEFs. The induced p53 was capable of sequence-specific DNA binding even in the absence of DNA-PKcs. Transactivation of the cyclin-dependent-kinase inhibitor p21, a downstream target of p53, and the G1 cell cycle checkpoint were also found to be normal in the DNA-PKcs −/− MEFs. Our results demonstrate that DNA-PKcs, unlike the related ATM protein, is not essential for the activation of p53 and G1cell cycle arrest in response to ionizing radiation.

Mouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus

We identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN.

ERK signaling controls blastema cell differentiation during planarian regeneration

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.

MURINE CELL LINE SX9 BEARING A MUTATION IN THE DNA-PKCS GENE EXHIBITS ABERRANT V(D)J RECOMBINATION NOT ONLY IN THE CODING JOINT BUT ALSO IN THE SIGNAL JOINT

We established the radiosensitive cell line SX9 from mammary carcinoma cell line FM3A. In SX9 cells a defect of DNA-dependent protein kinase (DNA-PK) activity was suggested. Additionally, a complementation test suggested that the SX9 cell line belongs to a x-ray cross-complementing group (XRCC) 7. Isolation and sequence analyses of DNA-dependent protein kinase catalytic subunit (dna-pkcs) cDNA in SX9 cells disclosed nucleotide “T” (9572) to “C” transition causing substitution of amino acid residue leucine (3191) to proline. Interestingly, the mutation occurs in one allele, and transcripts of the dna-pkcs expressed exclusively from mutated allele. V(D)J recombination assay using extrachromosomal vector revealed the defects of not only coding but also signal joint formation. The frequency of the signal joint decreased to approximately one-tenth and the fidelity drastically decreased to 12.2% as compared with the normal cell line. To confirm the responsibility of thedna-pkcs gene for abnormal V(D)J recombination in SX9, the full-length dna-pkcs gene was introduced into SX9. As a result, restoration of V(D)J recombination by wild typedna-pkcs cDNA was observed. SX9 is a noveldna-pkcs-deficient cell line.

Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro

Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.

Crucial role of c-Myc in the generation of induced pluripotent stem cells.

c‐Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c‐Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration‐free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c‐Myc (4F‐iPSCs) and those produced without c‐Myc (3F‐iPSCs). Molecular and cellular analyses did not reveal any differences between 3F‐iPSCs and 4F‐iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F‐iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F‐iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c‐Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F‐iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c‐Myc transduction for preparing high‐quality iPSCs. STEM CELLS 2011; 29:1362–1370

Comprehensive gene expression analyses in pluripotent stem cells of a planarian, Dugesia japonica

The neoblasts are the only somatic stem cells in planarians possessing pluripotency, and can give rise to all types of cells, including germline cells. Recently, accumulated knowledge about the transcriptome and expression dynamics of various pluripotent somatic stem cells has provided important opportunities to understand not only fundamental mechanisms of pluripotency, but also stemness across species at the molecular level. The neoblasts can easily be eliminated by radiation. Also, by using fluorescence activated cell sorting (FACS), we can purify and collect many neoblasts, enabling identification of neoblast-related genes by comparison of the gene expression level among intact and X-ray-irradiated animals, and purified neoblasts. In order to find such genes, here we employed the high coverage expression profiling (HiCEP) method, which enables us to observe and compare genome-wide gene expression levels between different samples without advance sequence information, in the planarian D. japonica as a model organism of pluripotent stem cell research. We compared expression levels of ~17,000 peaks corresponding to independent genes among different samples, and obtained 102 peaks as candidates. Expression analysis of genes identified from those peaks by in situ hybridization revealed that at least 42 genes were expressed in the neoblasts and in neoblast-related cells that had a different distribution pattern in the body than neoblasts. Also, single-cell PCR analysis of those genes revealed heterogeneous expression of some genes in the neoblast population. Thus, using multidimensional gene expression analyses, we were able to obtain a valuable data set of neoblast-related genes and their expression patterns.

Conversion of Ancestral Fibroblasts to Induced Pluripotent Stem Cells

The emergence of induced pluripotent stem cells (iPSCs) from an ancestral somatic cell is one of the most important processes underlying their generation, but the mechanism has yet to be identified. This is principally because these cells emerge at a low frequency, about 0.1% in the case of fibroblasts, and in a stochastic manner. In our current study, we succeeded in identifying ancestral fibroblasts and the subsequent processes leading to their conversion to iPSCs. The ancestral fibroblasts were found to divide several times in a morphologically symmetric manner, maintaining a fibroblastic shape, and then gradually transform into embryonic stem‐like cells. Interestingly, this conversion occurred within 48 hours after gene introduction in most iPSC generations. This is the first report to directly observe a cell lineage conversion of somatic cells to stem cells and provides a critical new insight into the “black box” of iPSCs, that is, the first three days of their generation. STEM CELLS 2010;28:213–220