Expression Of Early Transcription Factors Research Articles

Material matters: Degradation products affect regenerating Schwann cells

Devices to treat peripheral nerve injury (PNI) must balance many considerations to effectively guide regenerating nerves across a gap and achieve functional recovery. To enhance efficacy, design features like luminal fillers have been explored extensively. Material choice for PNI devices is also critical, as the determining factor of device mechanics, and degradation rate and has increasingly been found to directly impact biological response. This study investigated the ways in which synthetic polymer materials impact the differentiation state and myelination potential of Schwann cells, peripheral nerve glia. Microporous substrates of polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15, or PLGA 50:50 were chosen, as materials already used in nerve repair devices, representing a wide range of mechanics and degradation profiles. Schwann cells co-cultured with dorsal root ganglion (DRG) neurons on the substrates expressed more mature myelination proteins (MPZ) on PLGA substrates compared to PCL. Changes to myelination and differentiation state of glia were reflected in adhesion proteins expressed by glia, including β-dystroglycan and integrin α6, both laminin binding proteins. Importantly, degradation products of the polymers affected glial expression independently of direct attachment. Fast degrading PLGA 50:50 substrates released measurable amounts of degradation products (lactic acid) within the culture period, which may push Schwann cells towards glycolytic metabolism, decreasing expression of early transcription factors like sox10. This study shows the importance of understanding not only material effects on attachment, but also on cellular metabolism which drives myelination responses.

Expression of early transcription factors by mesenchymal stem cells derived from ovine umbilical cord Wharton’s jelly

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into mesodermal lineages. Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow MSCs has remained to be challenged. In this study, we utilized ovine umbilical cord Wharton’s jelly as a primary source for isolation of MSCs since it is a rich source of MSCs and no ethical issues were involved. Ovine umbilical cord Wharton’s jelly segments were digested enzymatically and cultured in vitro in culture medium. In addition to the study of their morphology and colony forming units, the expression of pluripotent stem cell markers by the isolated MSCs were also studied. The MSCs were plastic adherent, clonogenic and their morphology were polygonal, star shaped and fibroblast like. They revealed a strong expression of pluripotent stemness markers Oct4, Sox2, Nanog and Alkaline phosphatase. These cells confirmed their ability of self-renewal by expressing Sox2 gene and their properties of pluripotency and plasticity by expressing Oct4, Nanog and Alkaline phosphatase. The study revealed that Wharton’s jelly is a rich source of stem cells with stemness properties and mesenchymal like morphology and could be used as an alternate for the bone marrow derived MSCs for cell based regenerative therapies.

Sleeve gastrectomy activates the GLP-1 pathway in pancreatic β cells and promotes GLP-1-expressing cells differentiation in the intestinal tract

Db/db mouse was used to study the underlying mechanisms by which Sleeve gastrectomy (SG) improves β-cell function. We investigated β-cell function, plasma active GLP-1 levels, the GLP-1R pathway in β cells and L cell differentiation. After SG, β-cell function was significantly increased, and the GLP-1R-PKCζ-PDX-1 pathway was active in β cells. Plasma active GLP-1 levels, as well as the number of L cells in the jejunum, were significantly increased after SG. The expression of early transcription factors (TF), including Ngn3, FoxA1 and Nkx2.2, was not compromised by chronic hyperglycemia. In contrast, the expression of the downstream TF PAX6 was affected, and this down-regulation could be reversed by SG. So, SG can maintain L cell differentiation, increase plasma active GLP-1 level, sustain the activation of the GLP-1R pathway and improve β cell function in Db/db mice. Our results show that SG can overall improve the function of the entero-insular axis.

279 COMPARISONS OF BONE MARROW AND PUTATIVE SKIN-DERIVED MESENCHYMAL STEM CELLS AS DONOR FOR NUCLEAR TRANSFER IN MINIATURE PIG

Since the birth of the first cloned sheep was reported, fibroblasts are commonly used as donor cells for nuclear transfer. However, in some species there is a higher incidence of abnormal fetuses, still births, and neonatal deaths as a consequence of reprogramming disorders including DNA methylation and histone acetylation. Cloned embryos with mesenchymal stem cells (MSC) have shown higher developmental ability compared to fibroblast, suggesting that undifferentiated genome can required ease reprogramming. Because MSC are relatively difficult to collect from bone marrow, skin is an alternative source of the donor cells. However, molecular and functional analyses remain uncertain between MSC derived from bone marrow and skin stem cells isolated from ear. The present study compared the expression of early transcription factors (Oct-4, Nanog and Sox-2), and differentiation capability to osteocytes, adipocytes and chondrocytes of MSC isolated from bone marrow and skin-derived putative stem cells from miniature pig. Bone marrow was isolated by Ficoll density gradient method, and skin separated from epidermis and dermis was diced into 2-mm diameter explants, and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% FBS, 10 ng mL–1 bFGF, 10 ng mL–1 EGF at 38.5°C, in a humidified atmosphere of 5% CO2 in air. Expression of Oct-4, Nanog, Sox2 was analysed by RT-PCR. Osteogenic and adipogenic differentiation were induced following the protocols described previously (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells 24, 343–350) and compared by histological staining and RT-PCR. Osteocytes were defined by the formation of the mineral nodules of deposition of calcium by Von Kossa staining and differentiations into adipocytes and chondrocytes were identified by oil red O staining of lipid vacuoles and alcian blue of proteoglycan, respectively. Skin-derived MSC were revealed to similar mRNA expression of Oct-4, Nanog, Sox2 compared to bone marrow derived MSC. However, bone marrow derived MSC were higher mRNA expression about of osteocytic genes (osteoclacin and osteonectin), chondrocytic gene (collagen type) and adipocytic genes (aP2) than those of skin-derived MSC. In addition, bone marrow derived MSC were revealed greater deposition of calcium, proteoglycan, and lipid vacuoles than those of skin derived MSC by histological staining. The results of present study suggest that cells isolated from skin have lower potential than MSC isolated from bone marrow. However, skin-derived stem cells have properties of multi-lineage differentiation and can be obtained easily. These stem cells, therefore, can serve as easily accessible and expandable source possessing donor cells for cloning, potential animal model, and clinical applications. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.

283 EXPRESSION OF TRANSCRIPTION FACTORS AND MESENCHYMAL DIFFERENTIATION OF STEM CELLS DERIVED FROM WHARTON'S JELLY OF PORCINE UMBILICAL CORD

Wharton's jelly derived from mesoderm of umbilical cord matrix is an abundant source of stem cells such as mesenchymal and more primitive stem cells that don't raise the ethical issues associated with embryonic stem cells. The Oct-4 POU transcription factor, Nanog, and Sox-2 are expressed in embryonic stem cells and have been implicated in embryonic development and maintenance of pluripotency. The present study examined the expression of early transcription factors, Oct-4, Nanog, and Sox-2, and capability of differentiation to osteocytes, adipocytes, and chondrocytes of the cells of Wharton's jelly isolated from porcine umbilical cord. Wharton's jelly was obtained from the umbilical cord of post-natal piglets (immediately after birth). This tissue was diced into 2-mm-diameter explants and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% fetal blood serum (FBS), 10 ng mL–1 basic fibroblast growth factor (bFGF), 1 nm dexamethasone, and 10 nm insulin at 38.5�C, 5% CO2 in air. Expression of Oct-4, Nanog, and Sox-2 by these cells was analyzed by immunocytochemistry and RT-polymerase chain reaction (PCR), and compared with expression by porcine bone marrow MSCs established in our laboratory. Osteogenic, adipogenic, and chondrogenic differentiation were induced following the protocols described earlier (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells, in press). Cells isolated from Wharton's jelly expressed Oct-4, Nanog, and Sox-2 especially in colonies/clusters, as observed by immunocytochemistry and gene expression by RT-PCR, and this expression was higher than by MSCs isolated from bone marrow. Osteocytes were documented by the formation of the mineral nodules with alkaline phosphatase-positive cells and deposition of calcium. Differentiation into adipocytes was exhibited by oil red O staining of lipid vacuoles. Alcian blue staining for proteoglycans demonstrated the differentiation of chondrogenic cells. In conclusion, the results of the present study suggest that cells isolated from Wharton's jelly have properties of primitive pluripotency and multilineage differentiation. These stem cells, therefore, can serve as an easily accessible and expandable source possessing potential for preclinical applications using a large animal model. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.

Longitudinal Imaging Reveals Pituitary Enlargement Preceding Hypoplasia in Two Brothers with Combined Pituitary Hormone Deficiency Attributable to PROP1 Mutation

Mutations of the PROP-1 gene cause combined pituitary hormone deficiency. Progressive ACTH/cortisol insufficiency is found in a few patients. Congenital hypoplasia of the anterior pituitary gland is the most common magnetic resonance imaging finding in patients with PROP-1 mutations. We present two brothers with compound heterozygosity for the two mutations 150delA and 301-302delAG of the PROP-1 gene. Both showed combined pituitary hormone deficiency of GH, TSH, PRL, and gonadotropins, as is typical for PROP-1 deficiency. We observed a developing insufficiency of ACTH and cortisol secretory capacity in both patients. Computed tomography revealed an enlarged pituitary in the older brother at 3.5 yr of age. Repeated magnetic resonance imaging after 12 yr showed a constant hypoplasia of the anterior pituitary lobe. Similarly, magnetic resonance imaging of the younger brother showed a constant enlargement of the anterior pituitary gland until 10 yr. At the age of 11 yr, the anterior pituitary was hypoplastic. The reason for pituitary enlargement in early childhood with subsequent decrease in pituitary size is not known. We speculate that altered expression of early transcription factors could be involved. Because both patients have the same PROP-1 mutations and an identical pattern of combined pituitary hormone deficiency, we suggest that early pituitary enlargement may be the typical course in such patients in whom pituitary surgery is not indicated.

Differential effects of transforming growth factor-beta 1 and phorbol myristate acetate on cardiac fibroblasts. Regulation of fibrillar collagen mRNAs and expression of early transcription factors.

Cardiac fibroblasts are responsible for synthesis and deposition of fibrillar collagen types I and III. Transforming growth factor-beta 1 (TGF-beta 1) has been proved to increase collagen biosynthesis in various systems, both in vivo and in vitro. We have investigated the effect of TGF-beta 1 on collagen gene expression in cultured cardiac fibroblasts and have compared this effect with that of a mitogenic agent, phorbol myristate acetate (PMA). The regulation of collagen types I and III gene expression was examined by using cDNA probes to rat alpha 2 (I) and mouse alpha 1 (III) procollagens. Quiescent cultured cardiac fibroblasts from rabbit heart were treated with TGF-beta 1 (10-15 ng/ml) and PMA (200 ng/ml). After 24 hours of treatment with TGF-beta 1, the abundance of mRNA for pro-alpha 2 (I) and pro-alpha 1 (III) collagens was increased by 112% (p less than 0.001) and 97% (p = 0.05), respectively, in treated fibroblasts compared with untreated cells. However, PMA-treated cells showed an opposite response: a 42% (p = 0.01) decrease in mRNA levels for pro-alpha 2 (I) collagen was observed. Immunofluorescent staining of cardiac fibroblasts in culture with anti-type I collagen antibody showed that alterations in mRNA levels led to altered collagen synthesis: cellular collagen was relatively increased in TGF-beta 1-treated cells and significantly diminished in PMA-treated cells. The abundance of mRNA for pro-alpha 1 (III) collagen was not affected by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)