Comparison of Natural Killer Cells Differentiated from Various Pluripotent Stem Cells.

Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3-CD56+, and CD56- cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR).

Mitochondrial DNA mutations in extremely preterm infants with bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious chronic lung disease affecting extremely preterm infants. While mitochondrial dysfunction has been investigated in various medical conditions, limited research has explored mitochondrial DNA (mtDNA) gene mutations, specifically in BPD. This study aimed to evaluate mitochondrial mtDNA gene mutations in extremely preterm infants with BPD. In this prospective observational study, we enrolled a cohort of extremely preterm infants diagnosed with BPD. Clinical data were collected to provide comprehensive patient profiles. Peripheral blood mononuclear cells were isolated from whole-blood samples obtained within a defined timeframe. Subsequently, mtDNA extraction and sequencing using next-generation sequencing technology were performed to identify mtDNA gene mutations. Among the cohort of ten extremely preterm infants with BPD, mtDNA sequencing revealed the presence of mutations in seven patients, resulting in a total of twenty-one point mutations. Notably, many of these mutations were identified in loci associated with critical components of the respiratory chain complexes, vital for proper mitochondrial function and cellular energy production. This pilot study provides evidence of mtDNA point mutations in a subset of extremely preterm infants with BPD. These findings suggest a potential association between mitochondrial dysfunction and the pathogenesis of BPD. Further extensive investigations are warranted to unravel the mechanisms underlying mtDNA mutations in BPD.

Graphislactone A, a Fungal Antioxidant Metabolite, Reduces Lipogenesis and Protects against Diet-Induced Hepatic Steatosis in Mice.

Graphislactone A (GPA), a secondary metabolite derived from a mycobiont found in the lichens of the genus Graphis, exhibits antioxidant properties. However, the potential biological functions and therapeutic applications of GPA at the cellular and animal levels have not yet been investigated. In the present study, we explored the therapeutic potential of GPA in mitigating non-alcoholic fatty liver disease (NAFLD) and its underlying mechanisms through a series of experiments using various cell lines and animal models. GPA demonstrated antioxidant capacity on a par with that of vitamin C in cultured hepatocytes and reduced the inflammatory response induced by lipopolysaccharide in primary macrophages. However, in animal studies using an NAFLD mouse model, GPA had a milder impact on liver inflammation while markedly attenuating hepatic steatosis. This effect was confirmed in an animal model of early fatty liver disease without inflammation. Mechanistically, GPA inhibited lipogenesis rather than fat oxidation in cultured hepatocytes. Similarly, RNA sequencing data revealed intriguing associations between GPA and the adipogenic pathways during adipocyte differentiation. GPA effectively reduced lipid accumulation and suppressed lipogenic gene expression in AML12 hepatocytes and 3T3-L1 adipocytes. In summary, our study demonstrates the potential application of GPA to protect against hepatic steatosis in vivo and suggests a novel role for GPA as an underlying mechanism in lipogenesis, paving the way for future exploration of its therapeutic potential.

Selection of iPSCs without mtDNA deletion for autologous cell therapy in a patient with Pearson syndrome.

Screening for genetic defects in the cells should be examined for clinical application. The Pearson syndrome (PS) patient harbored nuclear mutations in the POLG and SSBP1 genes, which could induce systemic large-scale mitochondrial genome (mtDNA) deletion. We investigated iPSCs with mtDNA deletions in PS patient and whether deletion levels could be maintained during differentiation. The iPSC clones derived from skin fibroblasts (9% deletion) and blood mononuclear cells (24% deletion) were measured for mtDNA deletion levels. Of the 13 skin-derived iPSC clones, only 3 were found to be free of mtDNA deletions, whereas all blood-derived iPSC clones were found to be free of deletions. The iPSC clones with (27%) and without mtDNA deletion (0%) were selected and performed in vitro and in vivo differentiation, such as embryonic body (EB) and teratoma formation. After differentiation, the level of deletion was retained or increased in EBs (24%) or teratoma (45%) from deletion iPSC clone, while, the absence of deletions showed in all EBs and teratomas from deletion-free iPSC clones. These results demonstrated that non-deletion in iPSCs was maintained during in vitro and in vivo differentiation, even in the presence of nuclear mutations, suggesting that deletion-free iPSC clones could be candidates for autologous cell therapy in patients. [BMB Reports 2023; 56(8): 463-468].

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells.

BACKGROUND: Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. METHODS: We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. RESULTS: We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. CONCLUSION: This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

De Novo Development of mtDNA Deletion Due to Decreased POLG and SSBP1 Expression in Humans.

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.

Mitochondrial genome mutations in mesenchymal stem cells derived from human dental induced pluripotent stem cells.

Ethical and safety issues have rendered mesenchymal stem cells (MSCs) popular candidates in regenerative medicine, but their therapeutic capacity is lower than that of induced pluripotent stem cells (iPSCs). This study compared original, dental tissue-derived MSCs with re-differentiated MSCs from iPSCs (iPS-MSCs). CD marker expression in iPS-MSCs was similar to original MSCs. iPS-MSCs expressed higher in pluripotent genes, but lower levels in mesodermal genes than MSCs. In addition, iPS-MSCs did not form teratomas. All iPSCs carried mtDNA mutations; some shared with original MSCs and others not previously detected therein. Shared mutations were synonymous, while novel mutations were non-synonymous or located on RNA-encoding genes. iPS-MSCs also harbored mtDNA mutations transmitted from iPSCs. Selected iPS-MSCs displayed lower mitochondrial respiration than original MSCs. In conclusion, screening for mtDNA mutations in iPSC lines for iPS-MSCs can identify mutation-free cell lines for therapeutic applications. [BMB Reports 2019; 52(12): 689-694].

Stem cells and reproduction.

Reproductive biotechnology has developed rapidly and is now able to overcome many birth difficulties due to infertility or the transmission of genetic diseases. Here we introduce the next generation of assisted reproductive technologies (ART), such as mitochondrial replacement technique (MRT) or genetic correction in eggs with micromanipulation. Further, we suggest that the transmission of genetic information from somatic cells to subsequent generations without gametes should be useful for people who suffer from infertility or genetic diseases. Pluripotent stem cells (PSCs) can be converted into germ cells such as sperm or oocytes in the laboratory. Notably, germ cells derived from nuclear transfer embryonic stem cells (NT-ESCs) or induced pluripotent stem cells (iPSCs) inherit the full parental genome. The most important issue in this technique is the generation of a haploid chromosome from diploid somatic cells. We hereby examine current science and limitations underpinning these important developments and provide recommendations for moving forward. [BMB Reports 2019; 52(8): 482-489].

Functional Human Oocytes Generated by Transfer of Polar Body Genomes.

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Combination of soya pulp and Bacillus coagulans lilac-01 improves intestinal bile acid metabolism without impairing the effects of prebiotics in rats fed a cholic acid-supplemented diet.

Intestinal bacteria are involved in bile acid (BA) deconjugation and/or dehydroxylation and are responsible for the production of secondary BA. However, an increase in the production of secondary BA modulates the intestinal microbiota due to the bactericidal effects and promotes cancer risk in the liver and colon. The ingestion of Bacillus coagulans improves constipation via the activation of bowel movement to promote defaecation in humans, which may alter BA metabolism in the intestinal contents. BA secretion is promoted with high-fat diet consumption, and the ratio of cholic acid (CA):chenodeoxycholic acid in primary BA increases with ageing. The dietary supplementation of CA mimics the BA environment in diet-induced obesity and ageing. We investigated whether B. coagulans lilac-01 and soya pulp influence both BA metabolism and the maintenance of host health in CA-supplemented diet-fed rats. In CA-fed rats, soya pulp significantly increased the production of secondary BA such as deoxycholic acid and ω-muricholic acids, and soya pulp ingestion alleviated problems related to plasma adiponectin and gut permeability in rats fed the CA diet. The combination of B. coagulans and soya pulp successfully suppressed the increased production of secondary BA in CA-fed rats compared with soya pulp itself, without impairing the beneficial effects of soya pulp ingestion. In conclusion, it is possible that a combination of prebiotics and probiotics can be used to avoid an unnecessary increase in the production of secondary BA in the large intestine without impairing the beneficial functions of prebiotics.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BACKGROUND: Recent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine. METHODS: To enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis. RESULTS: Ovarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice. CONCLUSIONS: These findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source.

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated ß-galactosidase (SA-ß-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFß, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-ß-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue.

Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] ß and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFß and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Development and gene expression of porcine cloned embryos derived from bone marrow stem cells with overexpressing Oct4 and Sox2.

The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors.

Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis.

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

Ovarian-cell-like cells from skin stem cells restored estradiol production and estrus cycling in ovariectomized mice.

Reduction of estradiol production and high serum concentrations of follicular stimulating hormone (FSH) are endocrine disorders associated with premature ovarian failure. Here, we report that transplantation of ovarian-like cells differentiated from stem cells restored endogenous serum estradiol levels. Stem cells were isolated from postnatal mouse skin and differentiated into ovarian-cell-like cells that are consistent with female germ, and ovarian follicle somatic cells. The ovarian-cell-like cells were transplanted into ovariectomized mice (Cell Trans), whereas control mice were subjected to bilateral ovariectomies without cell transplantation (OVX). Using vaginal cytology analysis, it was revealed that in 13 out of 19 Cell Trans mice, estrus cycles were restored around 8 weeks after cell transplantation and were maintained until 16 weeks post-transplantation, whereas in the OVX group, all mice were arrested at metestrus/diestrus of the estrus cycle. The uterine weight in the Cell Trans group was similar to sham operation mice (Sham OP), while severe uterine atrophy and a decreased uterine weight were observed in the OVX group. Histologically, ectopic follicle-like structures and blood vessels were found within and around the transplants. At 12-14 weeks after cell transplantation, mean serum estradiol level in Cell Trans mice (178.0±35 pg/mL) was comparable to that of the Sham OP group (188.9±29 pg/mL), whereas it was lower in the OVX group (59.0±4 pg/mL). Serum FSH concentration increased in the OVX group (1.62±0.32 ng/mL) compared with the Sham OP group (0.39±0.34 ng/mL). Cell Trans mice had a similar FSH level (0.94±0.23 ng/mL; P<0.05) to Sham OP mice. Our results suggest that ovarian somatic cells differentiated from stem cells are functional in vivo. In addition to providing insights into the function of ovarian somatic cells derived from stem cells, our study may offer potential therapeutic means for patients with hypo-estradiol levels like those encountered in premature ovarian failure.

NMR investigation on the DNA binding and B-Z transition pathway of the Zα domain of human ADAR1.

Human ADAR1, which has two left-handed Z-DNA binding domains, preferentially binds Z-DNA rather than B-DNA with a high binding affinity. Z-DNA can be induced in long genomic DNA by Z-DNA binding proteins through the formation of two B-Z junctions with the extrusion of one base pair from each junction. We performed NMR experiments on complexes of Zα(ADAR1) with three DNA duplexes at a variety of protein-to-DNA molar ratios. This study confirmed that the Zα(ADAR1) first binds to an 8-bp CG-rich DNA segment via a unique conformation during B-Z transition and the neighboring AT-rich region becomes destabilized. We also found that, when DNA duplexes have only 6-bp CG-rich segment, the interaction with Zα(ADAR1) did not affect the thermal stabilities of the 6-bp CG-rich segment as well as the neighboring two A·T base pairs. These results indicate that four Zα(ADAR1) proteins interact with the 8-bp DNA sequence containing a 6-bp CG-repeat segment as well as a dinucleotide step, even though the dinucleotid step contains A∙T base pairs. Thus this study suggests that the length of the CG-rich region is more important than the specific DNA sequence for determining which base-pair is extruded from the B-Z junction structure. This study also found that the Zα(ADAR1) in complex with a 11-bp DNA duplex exhibits a Z-DNA-bound conformation distinct from that of free Zα(ADAR1) and the initial contact conformations of Zα(ADAR1) complexed with 13-bp DNA duplexes.

Transplantation of porcine umbilical cord matrix mesenchymal stem cells in a mouse model of Parkinson's disease.

The present study compared mesenchymal stem cells derived from umbilical cord matrix (UCM-MSCs) with bone marrow (BM-MSCs) of miniature pigs on their phenotypic profiles and ability to differentiate in vitro into osteocytes, adipocytes and neuron-like cells. This study further evaluated the therapeutic potential of UCM-MSCs in a mouse Parkinson's disease (PD) model. Differences in expression of some cell surface and cytoplasm specific markers were evident between UCM-MSCs and BM-MSCs. However, the expression profile indicated the primitive nature of UCM-MSCs, along with their less or non-immunogenic features, compared with BM-MSCs. In vitro differentiation results showed that BM-MSCs had a higher tendency to form osteocytes and adipocytes, whereas UCM-MSCs possessed an increased potential to transform into immature or mature neuron-like cells. Based on these findings, UCM-MSCs were transplanted into the right substantia nigra (SN) of a mouse PD model. Transplantation of UCM-MSCs partially recovered the mouse PD model by showing an improvement in basic motor behaviour, as assessed by rotarod and bridge tests. These observations were further supported by the expression of markers, including nestin, tyrosine hydroxylase (TH), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), at the site of cell transplantation. Our findings of xenotransplantation have collectively suggested the potential utility of UCM-MSCs in developing viable therapeutic strategies for PD.