Se-Pill Park

Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.

The aim of this study was to produce dopaminergic neurons in vitro from human embryonic stem (hES) cells following treatment of various neurotrophic factors. MB03 hES cells were induced by retinoic acid (RA) or basic fibroblast growth factor (bFGF), which were further treated with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)-alpha in each induction method during neuron differentiation days. At the final differentiation stage (21 days), all treatment groups revealed very similar levels (bFGF, 76-78%; RA, 70-74%) of mature neurons (anti-NF-200) in two induction methods irrespective of the addition of BDNF or TGF-alpha. In addition, immunostaining and HPLC analyses revealed higher levels of tyrosine hydroxylase (20+/-2.3%) and dopamine (265.5+/-62.8 pg/ml) in the bFGF- and TGF-alpha-treated hES cells than in RA- or BDNF-treated hES cells. These data are one of the first reports on the generation of dopaminergic neurons of hES cells in vitro. Also, our results indicate that TGF-alpha may be successfully used in the bFGF induction protocol and yield higher numbers of dopaminergic neurons from hES cells.

Effect of human embryonic stem cell-derived neuronal precursor cell transplantation into the cerebral infarct model of rat with exercise

We analyzed the therapeutic effect of the transplantation of the human embryonic stem cell (NIH Code: MB01)-derived neuronal precursor (hES-NP) cell and post-ischemic exercise in rats with the middle cerebral artery (MCA) infarct model. A cortical infarct was induced in 20 adult Sprague-Dawley rats by occlusion and reperfusion of the MCA. The rats were divided into four groups: hES-NP cell transplantation and exercise, transplantation only, exercise only, and Sham-operated with no exercise. In the cell-transplanted group, hES-NP cells were transplanted by stereotactic inoculation into the ipsilateral basal ganglia 7 days after infarct. We evaluated the clinical recovery of deficit, the size of infarct and the survival, migration, and differentiation of the transplanted cells. The transplanted hES-NP cells survived robustly in the ischemic brains 3 weeks post transplant. The majority of migrating cells in the ischemic rats had a neuronal phenotype. The clinical scores of all of the experimental groups were better than those of the Sham-operated group. Whereas the exercise-only group showed continuous clinical improvement, the cell-transplanted groups manifested less improvement than the exercise-only group. Moreover, the cell-transplanted groups did not differ in clinical improvement according to postinfarct-exercise or not. The infarct size was significantly reduced in both the cell-transplanted groups and the post-ischemic exercise group, compared with the Sham-operated group; however, the reduction of infarct size was most prominent in the exercise-only group. In our study, the inoculated site of the basal ganglia showed some damage induced by inoculation, such as loss of neuroglial cells, reactive gliosis and microcalcification, which was found in the Sham-operated group as well, and yet no inoculation-site injury has ever been reported. Our study revealed that stem cell transplantation can have a positive effect on behavioral recovery and reduction of infarct size, but the effect shown was no better than the effect of the exercise, which finding reconfirmed the importance of post-infarct rehabilitation. In addition, it was found that cell inoculation should be replaced by a noninvasive procedure.

Genetically modified human embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson's disease

Embryonic stem (ES) cells have great potential as a cell source for cell replacement therapy. To investigate the possibility of using ES cells as a carrier of therapeutic gene(s), human ES cells (MB03) were co-transfected with cDNAs coding for tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH I), then bulk-selected in the presence of neomycin and hygromycin-B. Successful transfection was confirmed by Western immunoblotting and RT-PCR. The genetically modified ES cells (bk-THGC) were found to produce a significant amount of L-dopa spontaneously and relieved apomorphine-induced asymmetric motor behavior by approximately 54% when grafted into striatum of 6-OHDA-denervated rat brain. The number of rotations, however, increased up to 176+/-18% in 6 weeks when PBS was used instead (sham-graft). Immunohistochemical stainings revealed that the grafted human ES cells survived and expressed TH for at least 6 weeks while the experiment was continued.

Enhancement of re-closure capacity by the intra-amniotic injection of human embryonic stem cells in surgically induced spinal open neural tube defects in chick embryos.

To evaluate the re-closure promoting capacity of human embryonic stem (hES) cells injected into the amniotic cavity on spinal open neural tube defects (ONTDs) of chick embryos, neural tubes were opened at Hamburger and Hamilton stage 18 or 19 and the embryos were divided into three groups: a control group (no injection), a vehicle group, and a hES cell group (injection of 20,000 hES cells immediately after neural tube incision). On postoperative days 3, 5, and 7, ONTDs were significantly more re-closed in the hES cell group than in the other two groups. hES cells were present at the area in the process of re-closure, and covered ONTDs, but were not found in the re-closed area, suggesting indirect effects rather than cell replacement on the neural tissue.

Enhanced Hatching Rate of Bovine IVM/IVF/IVC Blastocysts Using a 1.48-μm Diode Laser Beam

Purpose:Our purpose was to test whether zona pellucida (ZP) drilling using a 1.48-μm diode laser beam on bovine IVM/IVF/IVC blastocysts is effective for embryo hatching.Methods:Blastocysts produced in vitro at day 7 after IVF were divided into control and laser-drilled groups, respectively.Results:When the rates of in vitro development of bovine embryos were examined, the average cleavage rate (>two-cell) was 82.3% and the blastocyst rate at day 7 after IVF was 32.5%. Using these blastocysts, when the laser drilling effect was investigated at 48 hr after treatment, the total hatching rate in the laser-drilled group (98.0%) was significantly higher than that in the control group (60.0%)(P < 0.001). Especially, the hatched rate of the laser-drilled group (68.0%) was significantly enhanced compared with that of the control group (30.0%) (P < 0.001).Conclusions:These results demonstrated that laser ZP drilling on bovine IVM/IVF/IVC blastocysts can significantly increase the hatching rate.

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development

Human adipose tissue‐derived mesenchymal stem cells (hAT‐MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT‐MSC bioactive material (hAT‐MSC‐BM), and examined the effect of hAT‐MSC‐BM on porcine embryo development. hAT‐MSC‐BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT‐MSC‐BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT‐MSC culture medium group, the best results were from the group cultured with 10% hAT‐MSC‐BM. Mitochondrial activity was also higher in the 10% hAT‐MSC‐BM‐treated group. Moreover, the relative mRNA expression levels of development and anti‐apoptosis genes were significantly higher in the 10% hAT‐MSC‐BM‐treated group than in control, hAT‐MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT‐MSC‐BM starting on Day 4 also improved the development rate and the total cell number of in vitro‐fertilized embryos. This is the first report on the benefits of hAT‐MSC‐BM in a porcine embryo in vitro culture system. We conclude that hAT‐MSC‐BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro. Mol. Reprod. Dev. 80: 1035–1047, 2013.

Coat Color Patterns and Genotypes of Extension and Agouti in Hanwoo and Jeju Black Cattle

To understand the relationship between coat color inheritance patterns and genotypes of Extension( E) and Agouti( A) loci in cattle, the genotypes for melanocortin-1 receptor( MC1R) and agouti signaling protein ( ASIP) were analyzed in Hanwoo, Jeju black cattle (JBC), and their crossbred progeny. Three MC1R alleles ( E D , E?, and e) were found in the black-colored JBC population. JBC had no recessive homoygotes (e/e), but this genotype was predominant in the Hanwoo breed. However, MC1R E?/eHanwoo did not produce a black coat color as they appeared either as brown or solid red. For ASIP, three genotypes ( A/A, A/A Br , and A Br /A Br ) were determined by insertion/deletion of an L1-BT element in Hanwoo. The ASIP A Br allele was rarely observed, and no ASIP A Br /A Br homozygotes were detected in the JBC population. Cattle carrying ASIP ABr did not show any agouti-like brindle pigmentation patterns in either breed or their progeny. The coat colors of the crossbred progeny were discriminated by two colors, yellowish-brown versus dark-brown or black, and their coat colors were directly related to the genotypes of the Extensionlocus, yellowish-brown (e/e) and dark-brown or black (E?/e), but not to the Agouti locus. ASIP genotypes probably did not affect coat color development in the Hanwoo or crossbred progeny. Our results suggest that the ASIP genotypes do not play key roles in coat color variation, but the MC1R genotypes do direct the phenotypes of Hanwoo, JBC, and their progeny.

Production of transgenic pig as an Alzheimer’s disease model using a multi-cistronic vector system

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2–1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aβ-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes†

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus‐oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocytes metaphase‐II (MII) stage was significantly higher in the 10F‐treated group than in the control. Transcript abundance of the cumulus cell expansion‐related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F‐ and 50F‐treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F‐treated group. The percentage of oocytes with normal spindles was greater in the 10F‐ and 50F‐treated group at GVBD than in the other groups; the 5F‐, 10F‐, and 100F‐treated groups were higher than the control; and the 50F‐treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F‐treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F‐ to 50F‐treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67–75, 2017.

Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/–) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/– (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/– (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.