Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

Muscular satellite cell (SC), which is stem cell of postnatal pig, is an important for study of differentiation into adipogenesis, myogenesis, and osteoblastogenesis. In this study, we isolated and examined from pig muscle tissue to determine capacity in proliferate, differentiate, and expression of various genes. Porcine satellite cells (PSC) were isolated from semimembranosus (SM) muscles of 90∼100 days old pigs according to standard conditions. The cell proliferation increased in multi-potent cell by Masson’s, oil red O, and Alizarin red staining respectively. We per-formed the expression levels of differentiation related genes using real-time PCR. We found that the differentiation into adipocyte increased expression levels of both fatty acid binding protein 4 (FABP4) and peroxisome proliferator- acti-vated receptor gamma (PPARγ) genes (p<0.01). Myocyte increased the expression levels of the myosin heavy chain (MHC), myogenic factor 5 (Myf5), myogenic regulatory factor (MyoD), and Myogenic factor 4 (myogenin) (p<0.01). Osteo-blast increased the expression levels of alkaline phosphatase (ALP) (p<0.01). Finally, porcine satellite cells were indu-ced to differentiate towards adipogenic, myogenic, and osteoblastogenic lineages. Our results suggest that muscle satellite cell in porcine may influence cell fate. Understanding the progression of PSC may lead to improved strat-egies for augmenting meat quality.

Human dental pulp stem cells (DPSCs) are multi-potent mesenchymal stem cells that have several differentiation potentials. An understanding of thetissues that differentiate from these cells can provide insights for future regenerative therapeutics and tissue engineering strategies. The mesiodens is the most frequent form of supernumerary tooth from which DPSCs can differentiate into several lineages similar to cells from normal deciduous teeth. Recently, it has been shown that nanoscale structures can affect stem cell differentiation. In our presentstudy, we investigated the effects of a 250-nm nanoscale ridge/groove pattern array on the osteogenic and adipogenic differentiation of dental pulp cells from mesiodenscontaining human DPSCs. To this end, the expression of lineage specific markers after differentiation induction was analyzed by lineage specific staining and RT-PCR. The nanoscale pattern arrayed surface showed apositive effect on the adipogenic differentiation of DPSCs. There was no difference between nanoscale pattern arrayed surface and conventional surface groups onosteogenic differentiation. In conclusion, the nanoscale ridge/groove pattern arrayed surface can be used to enhance the adipogenic differentiation of DPSCs derived from mesiodens. This finding provides an improved understanding of the effects of topography on cell differentiation as well as the potential use of supernumerary tooth in regenerative dental medicine.

Insects possess two distinct acetylcholinesterases (AChE1 vs. AChE2), which are encoded by two paralogous loci originated from duplication. Kinetic analyses of several insect AChEs revealed that both AChE1 and AChE2 retain common catalytic properties of AChE but subtle kinetic differences also exist between these two AChEs. To understand how selection pressure has shaped the protein structure of AChEs and affected their function during evolution, we measured and compared the nucleotide diversity (Pi) and amino acid site-specific selection pressure between AChE1 and AChE2 from various insects. Highly conserved were the majority of the amino acid residues involved in forming the essential domains, including peripheral anionic site (PAS), and little differences were revealed between AChE1 and AChE2, suggesting the presence of strong purifying selection pressure over these essential residues. Interestingly, the EF-hand like motif was mostly found in the AChE1 lineage but not in AChE2. In addition, a unique amino acid difference in the PAS (D72 vs. Y72) was highly conserved between AChE1 and AChE2. Three-dimensional modeling of insect AChEs by particularly focusing on the PAS revealed that a subtle but consistent structural alteration in the active site topology was caused by the PAS amino acid substitution. Taken together, despite the long evolutionary history and low overall sequence similarity, both insect AChE1 and AChE2 still share a extremely high degree of structural and functional conservation, indicative of a strong purifying selection pressure. Nevertheless, only a small change in the PAS, appears to be associated with a local but significant alteration of AChE2 structure, which in turn drives the functional differentiation of AChE.

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation–mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchy-mal stem cells (hMSCs) and elucidate the underlying mole-cular mechanisms. Study design. The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1α and vas-cular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT- PCR analysis of osteogenic gene expression, an alkaline phos-phatase (ALP) activity assay and by alizarin red S staining. Results. Variable CoCl2 dosages (up to 500 µM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 µM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocal-cin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP acti-vity was increased in these treated cells in which an accele-rated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation poten-tial of hMSCs could be preserved and even enhanced by CoCl2 treatment.

This study investigated the genes involved in the dif- ferentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sor- ting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-poly- me rase chain reac tion (DD-PCR) was pe rformed to s c re en differentially expressed genes involved in the differentia- tion of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as β- glycerophosphate (β-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induc- tion. RT-PCR analysis revealed that dentin sialophospho- protein (DSPP) and osteocalcin (OC) expression had inc- reased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were diffe- rentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Further-more, the characterization of these regulated genes during odontogenic induction will likely provide valuable new in- sights into the functions of odontoblasts.

Recently, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. But there has been rarely reported about rehabilitaton of oral and maxillofacial bone defect using self-derived osteoblasts. Normal human osteoblast cell(NHost) was previously established into marrow-derived human mesenchymal stem cells for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. The purpose of this study was to examine proliferation and differentiation of NHosts effected by growth factors with ALP activity and RT-PCR. After NHosts were cultured under basal and osteogenic medium at 37℃ and 5% CO2, they were analyzed by ALP activity and RT-PCR. BMP-2 under osteogenic medium decreased growth rate of NHosts compared to under osteogenic medium. BMP-2 under osteogenic medium induced osteoblastic differentiation in NHosts by increased ALP activity. The differentiating capacity of NHosts under osteogenic medium showed that NHosts expressed higher mRNA expression levels of OSX and OCN, while that of RUNX2 decreased after BMP-2 treatment. It suggested that NHosts having characteristics of osteoprecursor cells might be more advanced in their osteogenesis development by BMP-2, making NHosts an interesting biological tool for treatment of skeletal defects and diseases of oral and maxillofacial bone.

MicroRNAs (miRNAs, miRs) are about 21-25 nucleotides in length and regulate mRNA translation by base pairing to partially complementary sites, predominantly in the 3’-untranslated region (3’-UTR) of the target mRNA. In this study, the expression profile of miRNAs was compared and analyzed for the establishment of miRNA-related odontoblast differentiation using MDPC-23 cells derived from mouse dental papilla cells. To determine the expression profile of miRNAs during the differentiation of MDPC-23 cells, we employed miRNA microarray analysis, quantitative real-time PCR (qRT-PCR) and Alizaline red-S staining. In the miRNA microarray analysis, 11 miRNAs were found to be up- or down-regulated more than 3-fold between day 0 (control) and day 5 of MDPC-23 cell differentiation among the 1,769 miRNAs examined. In qRT-PCR analysis, the expression levels of two of these molecules, miR-194 and miR-126, were increased and decreased in the control MDPC-23 cells compared with the MDPC-23 cells at day 5 of differentiation, respectively. Importantly, the overexpression of miR-194 significantly accelerated mineralization compared with the control cultures during the differentiation of MDPC-23 cells. These results suggest that the miR-194 augments MDPC-23 cell differentiation, and potently accelerates the mineralization process. Moreover, these in vitro results show that different miRNAs are deregulated during the differentiation of MDPC-23 cells, suggesting the involvement of these genes in the differentiation and mineralization of odontoblasts.

The synovial tissues are a valuable MSCs source for cartilage tissue engineering because these cells are easily obtainable by the intra-articular biopsy during diagnosis. In this study, we isolated and characterized the canine MSCs derived from synovial fluid of female and male donors. Synovial fluid was flushed with saline solution from pre and post-puberty male (cM1-sMSC and cM2-sMSC) and female (cF1-sMSC and cF2-sMSC) dogs, and cells were isolated and cultured in advanced-DMEM (A-DMEM) supplemented with 10% FBS in a humidified 5% atmosphere at . The cells were evaluated for the expression of the early transcriptional factors, such as Oct3/4, Nanog and Sox2 by RT-PCR. The cells were induced under conditions conductive for adipogenic, osteogenic, and chondrogenic lineages, then evaluated by specific staining (Oil red O, von Kossa, and Alcian Blue staining, respectively) and analyzed for lineage specific markers by RT-PCR. All cell types were positive for alkaline phosphatase (AP) activity and early transcriptional factors (Oct3/4 and Sox2) were also positively detected. However, Nanog were not positively detected in all cells. Further, these MSCs were observed to differentiate into mesenchymal lineages, such as adipocytes (Oil red O staining), osteocytes (von Kossa staining), and chondrocytes (Alcian Blue staining) by cell specific staining. Lineage-specific genes (osteocyte; osteonectin and Runx2, adipocytes; PRAR-, FABP and LEP, and chondrocytes; collagen type-2 and Sox9) were also detected in all cells. In this study, we successfully established synovial fluid derived mesenchymal stem cells from female and male dogs, and determined their basic biological properties and differentiation ability. These results suggested that synovial fluid is a valuable stem cell source for cartilage regeneration therapy, and it is easily accessible from osteoarthritic knee.

잉어목(Cypriniformes) 황어아과(Leuciscinae)의 황어(Tribolodon hakonensis)는 회유성 어류로서 일생의 대부분을 바다에서 보내고 산란기인 3월 중순경부터 물이 맑은 하천으로 소상하여 자갈이나 모랫바닥에 집단으로 알을 낳는다. 본 연구의 목적은 5개의 microsatellite 유전자 분석을 통하여 단편화된 하천에서 황어 집단 간 유전자 흐름과 다양성을 측정하는 것이다. 강원도 삼척 오십천은 여러 대형 보에 의해 부분적으로 단편화되어 있는 중형 하천으로, 본 연구에서 하류지역과 대형 보를 여러 번 지나야 다다를 수 있는 상류지역에서 채집한 황어 개체들의 유전자형을 비교, 분석하였다. 유전자 분석 결과 상, 하류 집단들은 많은 대립인자를 공유하지만 그 빈도에 있어 다소 큰 차이를 보였다. 상류와 하류 간 유전적 분화(FST)는 0.083 정도로 두 집단 간에는 제한된 유전적 흐름만이 존재한다고 볼 수 있다. 상류집단이 유전적으로 고립이 되어 있지만 뚜렷한 유전적 다양성의 감소나 집단의 크기 감소가 관찰되지는 않았다. 이러한 양상을 개체 수준에서 증명하기 위해 Bayesian 통계를 이용, 집단의 유전적 구조를 파악하였다. 분석 결과 삼척 오십천 개체들은 2개의 유전적 cluster로 구분할 수 있으며, 상류 집단 개체들은 모두 cluster 1에 해당하는 등 단일하게 나타났으나 하류 집단 개체 중 65 % 정도가 cluster 2에 그리고 나머지 개체들은 cluster 1에 해당하는 다양한 양상이 나타났다. 이로 미루어 두 집단은 유전적으로 분화되어 있고, 상류의 집단이 하류에 흘러들어가는 경우는 있지만 하류로부터 유전적 공급은 거의 전무한 형태로 볼 수 있고, 인위적 구조물들이 이러한 집단 구조에 영향을 미쳤을 가능성이 있다. 본 연구에서 제시된 자료들은 향후 황어 집단의 보전 정책 등을 수립하는데 필요한 정보를 제시할 수 있을 것이다.

Induced pluripotent stem cells (iPSCs), generated by the overexpression of transcription factors Oct4, Sox2, Klf4 and c‐Myc in somatic cells, are pluripotent. iPSCs reprogrammed from differentiated cells get through a epigenetic modification during reprogramming and finally have the similar epigenetic state to embryonic stem cells (ESCs). In this study, these epigenetic changes were observed in reprogramming of uni‐parental parthenogenetic somatic cells. Furthermore, we have shown that parthenogenetic pattern of imprinted genes were changed during pluripotential reprogramming. Parthenogenetic neural stem cells (pNSCs) containing only maternal alleles regain the biparental imprinting patterns after reprogramming. However, we have yet to define whether the changed imprinted genes are maintained or reverted to the parthenogenetic state when the reprogrammed cells are differentiated into specialized cell types. To address this question, we compared genome‐wide expression profiles of biparental female neural stem cells (fNSCs), parthenogenetic neural stem cells (pNSCs), and NSCs differentiated from parthenogenetic maternal iPSC (miPS‐NSCs). Furthermore, this study establishes the correlation between the alteration of genome methylation and activation of imprinting genes in the parthenogenetic cells and reports for the first time that the silenced PWS‐related imprinted genes are activated in miPS‐NSCs. Our data demonstrated that pluripotential reprogramming of parthenogenetic somatic cells were able to reset the parthenogenetic imprinting patterns; reprogrammed miPSCs showed erasure of maternal methylation imprints and acquisition of methylation in paternally imprinted genes. Furthermore, the changed imprinting patterns were maintained when the reprogrammed cells are differentiated into specialized cell type. * This work was supported by the Next‐Generation BioGreen 21 program (Grant PJ008- 009) funded by the Rural Development Administration, Republic of Korea.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

MAC-T cells, bovine mammary epithelial cell line, have been utilized to investigate bovine lactation system. A lactogenic phenotype of the cell is generally induced by combination of dexamethasone, insulin and prolactin (PRL). Effect of vitamin A derivative retinoic acid (RA), well reported as an inducer for differentiation in many cells, to MAC-T cell has not been studied. The objective of this study was to confirm effect of differentiation potential by RA treatment in MAC-T cells and to test effect of combination of RA and PRL treatment. In RA or PRL treatment groups, both has induced morphological change to secrete milk of MAC-T cells. Combination of RA and PRL treatment group has presented noticeable lactogenic phenotype among the all group. This phenotype observed at four days after treatment and showed critical morphological change that was rouphly spherical structure at eight days. RA alone treatment showed slightly inhibition of proliferation in the MAC-T cells, but co-treatment with PRL was improved the cell growth more than control group. MTT assay result and Bcl-xL/Bax ratio of mRNA abundance also was entirely consistent with earlier one. RA-induced differentiation of MAC-T cells has increased αs1-casein, αs2-casein and β-casein mRNA expression compared to PRL treatment group. Expression of αs1-casein, αs2-casein and β-casein genes represented the maximum value in the combination of RA and PRL treatment group at four days. The value of each casein gene expression was 4-, 5.5- and 5.9-fold, respectively, as compared with PRL alone treatment in the MAC-T cells. Protein level of β-casein releasing to the medium also induced the highest level at four days. These results provide evidence that RA can induce the differentiation of MAC-T cells and have synergetic effect with PRL.

Many polyphagous herbivore insects considered as a single species are indeed comprised of genetically differentiated host related races that utilize several unrelated plant families. Aphis gossypii Glover, one of the extremely polyphagous aphid species, has more than 100 host plants distributed worldwide. In Korea, it takes holocyclic life cycle, with a number of primary hosts, which is unusual for other aphid species. However, former population genetic studies on A. gossypii have dealt with the populations from only a few agricultural host plants. In this study, we hypothesized that different primary host utilization may be restricted for certain host races and this may play a key role of the genetic differentiation in this species. We collected the cotten aphid populations from 36 different host plants, 5 primary and 31 secondary hosts including various wild and non-cultivated plants. To determine population genetic structure, we analyzed genetic differentiation among those populations using 9 microsatellite loci. As results, four genetically distinct clusters were verified. Of the five primary host populations, there were significant gaps in genetic differences between Rhamnus-associated group and Hibiscus-associated group, and the other populations were affinitive with the Hibiscus-associated group. Overall, primary host populations have a higher level of genetic diversity than secondary host populations. Several populations associated with secondary hosts such as spiderwort, madder, and Korean lettuce were considerably differentiated in the result of principle component analysis, which nearly showed the species level difference similar to Aphis glycines Matsumura and Aphis sp. ex Rhamnus. Thus, there could be reproductively isolated by the loss of primary hosts, and might be already speciated from A. gossypii

Dlx3 and Dlx5 are homeobox domain proteins and are well-known regulators of osteoblastic differentiation. Since possible reciprocal relationships between osteogenic and adipogenic differentiation in mesenchymal stem cells exist, we examined the regulatory role of Dlx3 and Dlx5 on adipogenic differentiation using human dental pulp stem cells. Over-expression of Dlx3 and Dlx5 stimulated osteogenic differentiation but inhibited adipogenic differentiation of human dental pulp stem cells. Dlx3 and Dlx5 suppressed the expression of adipogenic marker genes such as C/EBPα, PPARγ, aP2 and lipoprotein lipase. Adipogenic stimuli suppressed the mRNA levels of Dlx3 and Dlx5, whereas osteogenic stimuli enhanced the expression of Dlx3 and Dlx5 in 3T3-L1 preadipocytes. These results suggest that Dlx3 and Dlx5 exert a stimulatory effect on osteogenic differentiation of stem cells through the inhibition of adipogenic differ¬entiation as well as direct stimulation.

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of Tuj1 increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous Igf2 may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders. Adipose tissue is ubiquitous and it can be easily harvested in large quantities under local anesthesia with little patient discomfort, making adipose tissue into the ideal large-scale source for research on clinical applications. In this study we monitored the neuronal cell differentiation potential of human adipocyte in the following condition; i) N2 medium containing 200 uM ascorbic acid (AA) and/or 10 uM flavonoid (F) and ⅱ) N2 medium containing AA and/or 10 ng/ml brain derived neurotrophic factor (BDNF) and/or, 200 ng/ml sonic hedgehog (SHH) plus 100 ng/ml fibroblast growth factor (FGF) 8. Adipose stem cells were cultured in above described differentiation condition for three weeks. RT-PCR analysis demonstrated that the mRNA levels of neuronal cell markers in differentiated adipose stem cells. Under the culture condition using N2 medium containing AA, the expression level of nestin (neural progenitor marker) m- RNA was high in all groups, while those of Neuro D, and LEP and FABP4 (adipocyte marker) mRNA were significantly decreased. Also, the addition of BDNF or SHH+FGF8 in N2 medium containing AA enhanced the neural cell differentiation from adipose stem cells, the expression level of Map2 (mature neuron) mRNA was increased, and that of TH (dopaminergic neuron marker) mRNA was high. In addition, we confirmed that the flavonoid addition has effect on the increase of Map2 expression. These results demonstrate that our designed culture condition has effect on the neural cell differentiation of adipose stem cells and this stimulatory effect may be further enhanced by transplantation.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

Somatic cells achieve a pluripotent state by pluripotential reprogramming. During pluripotential reprogramming, somatic cells re-established various features of pluripotent cells, such as the expression of pluripotency markers, inactivation of tissue-specific gene expression, developmental potential to contribute to all three germ layers, and an undifferentiated epigenetic state. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. These iPS cells are potentially a valuable source of immune matched pluripotent stem cells that can be differentiated and used for tissue replacement therapies. Recent technical advance in direct reprogramming of somatic cells lead to a safe, viral- free iPS cell generation. Here we develop new techniques to generate iPS cells. Using titanium oxide (TiO2) nanotubes. we could successfully transfer reprogramming proteins (Oct4, Sox2, Klf4, Nanog and c-Myc) into somatic cells. After two weeks of treatment of protein conjugated nanotubes, somatic cells adopted an ES cell-like morphology and activated Oct4-GFP, which is pluripotency marker, indicating that nanotubes can be used for protein delivery carriers, which induce cellular reprogramming. Next, we induced differentiation of iPS cells into neural stem cells (NSCs) and compared with mouse embryonic stem (ES) cell-derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from NSCs from brain tissue and rapidly propagated in the presence EGF and bFGF, and stained positive for NSCs markers Nestin and Sox2. Moreover, these NSCs have capacity of differentiation into multiple cell lineages, such as neurons, astrocytes, and oligodendrocytes. Induction of pluripotency and directed differentiation of iPS cells into a specialized cell type hold considerable promise for regenerative medicine as well as basic research.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.