The Efficiency of Neurospheres Derived from Human Wharton's Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats.

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and ß-tubulin 3 through the Wnt3A signaling pathway regulation markers (ß-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of ß-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-ß signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human.

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

The potential of the oleaginous yeast Rhodotorula paludigena CM33 to produce biolipids.

Sixty-seven yeast strains were isolated from castor beans then their endogenous lipids were stained by Nile Red (NR) fluorescence dye, and flow cytometry was used to obtain a strain with a high relative mean fluorescence intensity (MFI) value. The highest MFI value was obtained for strain CM33, which produced a maximum lipid content of 20.8 % dry cell weight (DCW). Based on the sequence of the ITS-5.8S-ITS rDNA and D1/D2 26S rDNA regions, CM33 showed 99 % identity with Rhodotorula paludigena. The potential of CM33 to assimilate various carbon sources was examined by growth on minimal media using glucose, glycerol, sucrose or xylose. CM33 was grown in glucose-based medium for 96 h and exhibited a maximum lipid content of 23.9 % DCW. Furthermore, when cells were cultured on molasses waste, their biomass, lipid content and lipid concentration reached 16.5 g/L, 37.1 % DCW and 6.1 g/L, respectively. These results demonstrated the potential of R. paludigena CM33 to contribute to a value-added carbon chain by converting renewable waste materials for biolipid production.

Differentiation Induction of Human Stem Cells for Corneal Epithelial Regeneration.

Deficiency of corneal epithelium causes vision impairment or blindness in severe cases. Transplantation of corneal epithelial cells is an effective treatment but the availability of the tissue source for those cells is inadequate. Stem cells can be induced to differentiate to corneal epithelial cells and used in the treatment. Multipotent stem cells (mesenchymal stem cells) and pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are promising cells to address the problem. Various protocols have been developed to induce differentiation of the stem cells into corneal epithelial cells. The feasibility and efficacy of both human stem cells and animal stem cells have been investigated for corneal epithelium regeneration. However, some physiological aspects of animal stem cells are different from those of human stem cells, the protocols suited for animal stem cells might not be suitable for human stem cells. Therefore, in this review, only the investigations of corneal epithelial differentiation of human stem cells are taken into account. The available protocols for inducing the differentiation of human stem cells into corneal epithelial cells are gathered and compared. Also, the pathways involving in the differentiation are provided to elucidate the relevant mechanisms.

Genome Sequence of the Oleaginous Yeast Rhodotorula paludigena Strain CM33, a Potential Strain for Biofuel Production.

The genome sequence of Rhodotorula paludigena strain CM33, an oleaginous yeast isolated from castor bean (Ricinus sp.) in Thailand, is reported here. Genome sequencing and assembly yielded 20,657,327 bases with a 64.3% G+C content.

Enhanced Hepatogenic Differentiation of Human Wharton's Jelly-Derived Mesenchymal Stem Cells by Using Three-Step Protocol.

Currently, human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an attractive source of stem cells for cell-based therapy, owing to their ability to undergo self-renewal and differentiate into all mesodermal, some neuroectodermal, and endodermal progenies, including hepatocytes. Herein, this study aimed to investigate the effects of sodium butyrate (NaBu), an epigenetic regulator that directly inhibits histone deacetylase, on hepatic endodermal lineage differentiation of hWJ-MSCs. NaBu, at 1 mM, optimally promoted endodermal differentiation of hWJ-MSCs, along with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) supplementation (EGF + bFGF + 1 mM NaBu). CXCR4, HNF3ß, SOX17 (endodermal), and GATA6 (mesendodermal) mRNAs were also up-regulated (p < 0.001). Immunocytochemistry and a Western blot analysis of SOX17 and HNF3ß confirmed that the EGF + bFGF + 1 mM NaBu condition was appropriately pre-treated with hWJ-MSCs before hepatogenic differentiation. Furthermore, the hepatogenic medium + NaBu pre-treatment up-regulated hepatoblast (AFP and HNF3ß) and hepatic (CK18 and ALB) markers, and increased the proportion of mature hepatocyte functions, including G6P, C/EBPα, and CYP2B6 mRNAs, glycogen storage and urea secretion. The hepatogenic medium + NaBu in the pre-treatment step can induce hWJ-MSC differentiation toward endodermal, hepatoblastic, and hepatic lineages. Therefore, the hepatogenic medium + NaBu pre-treatment for differentiating hWJ-MSCs could represent an alternative protocol for cell-based therapy and drug screening in clinical applications.

Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs) are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3) inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl) and SB216763 synergistically with TGF-ß3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs). hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and ß-catenin markers. Glycosaminoglycan (GAG) accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating ß-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.

Segregation of donor cell mitochondrial DNA in gaur-bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring.

The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur-bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT.

Full-term development of gaur-bovine interspecies somatic cell nuclear transfer embryos: effect of trichostatin A treatment.

Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development.

Development of intergeneric and intrageneric somatic cell nuclear transfer (SCNT) cat embryos and the determination of telomere length in cloned offspring.

Somatic cell nuclear transfer (SCNT) holds potential as a useful tool for agricultural and biomedical applications. In vitro development of marbled cat intergeneric SCNT reconstructed into domestic cat cytoplast revealed that cloned, marbled cat embryo development was blocked at the morula stage. No pregnancies resulted from the transfer of one- to eight-cell stage embryos into domestic cat surrogate mothers. This suggested that abnormalities occurred in the cloned marbled cat embryos, which may be associated with incomplete reprogramming during early embryo development. Two pregnancies were established in surrogate mothers that received cloned domestic cat embryos, but SCNT offspring developed abnormally. Some specific phenotypes that were observed included incomplete abdominal wall disclosure, improper fetal development. In addition, some of the fetuses were mummified or stillbirths. The two live births died within 5 days. Telomere lengths of cloned kittens as determined by qualtitative polymerase chain reaction (qPCR) were inconclusive: some were found to be shorter, longer, or the same as donor control cells. Our findings support the hypothesis that telomere lengths do not govern the health of these cloned animals. A lack of complete reprogramming may lead to developmental failure and the abnormalities observed in cloned offspring.

The effects of manipulation medium, culture system and recipient cytoplast on in vitro development of intraspecies and intergeneric felid embryos.

The aim of this study was to investigate if reconstructed felid embryos obtained by intraspecies or intergeneric cloning can develop in vitro. Fibroblast cells (f) from a domestic cat (DCf), marbled cat (MCf) and bovine (Bf) were used as donor cells, and oocytes (o) from domestic cats (DCo) and bovine (Bo) were used as recipient cytoplasts. There were two intraspecies (donor cell + recipient cytoplast: DCf + DCo and Bf + Bo) and three intergeneric (MCf + DCo, DCf + Bo and MCf + Bo) cloning groups in the study. In Experiment 1, the effects of manipulation media, modified TCM-199 (199H) or Emcare holding medium (EHM), on in vitro development of DCf + DCo embryos were investigated. The blastocyst formation rate (BFR) of the embryos manipulated in EHM (33.3%) was higher (P<0.05) compared with those manipulated in 199H (18.1%). In Experiment 2, DCf + DCo and MCf + DCo embryos were cocultured with or without domestic cat oviductal epithelium cells. Irrespective of coculture, the same BFR was obtained for DCf + DCo embryos (44.4 vs. 38.0%), while MCf + DCo embryos could not develop beyond the morula stage. In experiment 3, although the development of MCf + DCo and DCf + Bo embryos was arrested at the morula stage, 8.6% of MCf + Bo embryos were able to develop to the blastocyst stage. These results demonstrated that EHM was superior to 199H as an embryo manipulation medium and that the DCo and Bo could support the early embryonic development of intergeneric cloned marbled cat embryos up to the morula stage. However, postimplantation development still needs to be investigated.

A simple method for production and purification of soluble and biologically active recombinant human leukemia inhibitory factor (hLIF) fusion protein in Escherichia coli.

Mouse embryonic stem cells (mESCs) rely on a cytokine named leukemia inhibitory factor (LIF) to maintain their undifferentiated state and pluripotency. However, the progress of mESC research is restricted and limited to highly funded laboratories due to the cost of commercial LIF. Here we presented the homemade hLIF which is biologically active. The hLIF cDNA was cloned into two different vectors in order to produce N-terminal His6-tag and Trx-His6-tag hLIF fusion proteins in Origami(DE3) Escherichia coli. The His6-hLIF fusion protein was not as soluble as the Trx-His6-hLIF fusion protein. One-step immobilized metal affinity chromatography (IMAC) was done to recover high purity (> 95% pure) His6-hLIF and Trx-His6-hLIF fusion proteins with the yields of 100 and 200 mg/l of cell culture, respectively. The hLIF fusion proteins were identified by Western blot and verified by mass spectrometry (LC/MS/MS). The hLIF fusion proteins specifically promote the proliferation of TF-1 cells in a dose-dependent manner. They also demonstrate the potency to retain the morphology of undifferentiated mESCs, in that they were positive for mESC markers (Oct-4, Sox-2, Nanog, SSEA-1 and alkaline phosphatase activity). These results demonstrated that the N-terminal fusion tags of the His6-hLIF and Trx-His6-hLIF fusion proteins do not interfere with their biological activity. This expression and purification approach to produce recombinant hLIF is a simple, reliable, cost effective and user-friendly method.

Effect of donor cell types on developmental potential of cattle (Bos taurus) and swamp buffalo (Bubalus bubalis) cloned embryos.

This study investigated the effect of donor cell types on the developmental potential and quality of cloned swamp buffalo embryos in comparison with cloned cattle embryos. Fetal fibroblasts (FFs), ear fibroblasts (EFs), granulosa cells (GCs) and cumulus cells (CCs) were used as the donor cells in both buffalo and cattle. The cloned cattle or buffalo embryos were produced by fusion of the individual donor cells with enucleated cattle or buffalo oocytes, respectively. The reconstructed (cloned) embryos and in vitro matured oocytes without enucleation were parthenogenetically activated (PA) and cultured for 7 days. Their developmental ability to the blastocyst stage was evaluated. The total number of trophectoderm (TE) and inner cell mass (ICM) cells and the ICM ratio in each blastocyst was determined by differential staining as an indicator of embryo quality. The fusion rate of CCs with enucleated oocytes was significantly lower than for those of other donor cell types both in cattle and buffalo. The rates of cleavage and development to the 8-cell, morula and blastocyst stages of cloned embryos derived from all donor cell types did not significantly differ within the same species. However, the cleavage rate of cloned cattle embryos derived from FFs was significantly higher than those of cattle PA and cloned buffalo embryos. The blastocyst rates of cloned cattle embryos, except for the ones derived from CCs, were significantly higher than those of cloned buffalo embryos. In buffalo, only cloned embryos derived from CCs showed a significantly higher blastocyst rate than that of PA embryos. In contrast, all the cloned cattle embryos showed significantly higher blastocyst rates than that of PA embryos. There was no difference in ICM ratio among any of the blastocysts derived from any of the donor cell types and PA embryos in both species. FFs, EFs, GCs and CCs had similar potentials to support development of cloned cattle and buffalo embryos to the blastocyst stage with the same quality.