Duane L. Davis

Matrix cells from Wharton's jelly form neurons and glia.

We have identified an easily attainable source of primitive, potentially multipotent stem cells from Whartons jelly, the matrix of umbilical cord. Whartons jelly cells have been propagated in culture for more than 80 population doublings. Several markers for stem cells, including c‐kit (CD117), and telomerase activity are expressed in these cells. Treatment with basic fibroblast growth factor overnight and low‐serum media plus butylated hydroxyanisole and dimethylsulfoxide induced Whartons jelly cells to express a neural phenotype. Within several hours of this treatment, Whartons jelly cells developed rounded cell bodies with multiple neurite‐like extensions, similar to the morphology of neural stem cells. Neuron‐specific enolase (NSE), a neural stem cell marker, was expressed in these cells, as shown by immunocytochemistry. Immunoblot analysis showed similar levels of NSE expression in both untreated and induced Whartons jelly cells. After 3 days, the induced Whartons jelly cells resembled bipolar or multipolar neurons, with processes that formed networks reminiscent of primary cultures of neurons. The neuron‐like cells in these cultures stained positively for several neuronal proteins, including neuron‐specific class III β‐tubulin, neurofilament M, an axonal growth‐cone‐associated protein, and tyrosine hydroxylase. Immunoblot analysis showed increasing levels of protein markers for mature neurons over time postinduction. Markers for oligodendrocytes and astrocytes were also detected in Whartons jelly cells. These exciting findings show that cells from the matrix of umbilical cord have properties of stem cells and may, thus, be a rich source of primitive cells. This study shows their capacity to differentiate into a neural phenotype in vitro.

Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC) matrix cells

BackgroundThree transcription factors that are expressed at high levels in embryonic stem cells (ESCs) are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC) matrix cells.MethodsCells were isolated from Whartons jelly of porcine umbilical cords (PUC) and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells.ResultsPUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts.ConclusionCells isolated from PUC express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other characteristics of PUC cells such as their colony-forming ability, Hoechst dye-excluding side population and alkaline phosphatase expression, suggests that PUC cells have properties of primitive pluripotent stem cells. Furthermore, PUC cells are an easily and inexpensively obtained source of stem cells that are not hampered by the ethical or legal issues associated with ESCs. In addition, these cells can be cryogenically stored and expanded.

Prostaglandin E and F2α secretion by glandular and stromal cellsof the pig endometrium in vitro: Effects of estradiol-17β, progesterone, and day of pregnancy

Abstract Prostaglandins (PGs) are believed to play important roles in the establishment of pregnancy. Glandular and stromal cells were isolated from pig endometrium on days 11 through 19 of pregnancy and cultured in the presence of estradiol-17β (E2) and progesterone (P4) to determine the effect of day pregnancy and steroids on the secretion of PGE and PGF2α. Estradiol at concentrations between .01 and 1 μM did not affect PGE and PGF2α secretion into the medium by glandular and stromal cells. Progesterone (.1 μM) suppressed (P

Omega-3 fatty acids in the gravid pig uterus as affected by maternal supplementation with omega-3 fatty acids

Two experiments evaluated the ability of maternal fatty acid supplementation to alter conceptus and endometrial fatty acid composition. In Exp. 1, treatments were 1) the control, a corn-soybean meal diet; 2) flax, the control diet plus ground flax (3.75% of diet); and 3) protected fatty acids (PFA), the control plus a protected fish oil source rich in n-3 PUFA (Gromega, JBS United Inc., Sheridan, IN; 1.5% of diet). Supplements replaced equal parts of corn and soybean meal. When gilts reached 170 d of age, PG600 (PMSG and hCG, Intervet USA, Millsboro, DE) was injected to induce puberty, and dietary treatments (n = 8/treatment) were initiated. When detected in estrus, gilts were artificially inseminated. On d 40 to 43 of gestation, 7 gilts in the control treatment, 8 gilts in the PFA treatment, and 5 gilts in the flax treatment were pregnant and were slaughtered. Compared with the control treatment, the flax treatment tended to increase eicosapentaenoic acid (EPA: C20:5n-3) in fetuses (0.14 vs. 0.25 +/- 0.03 mg/g of dry tissue; P = 0.055), whereas gilts receiving PFA had more (P < 0.05) docosahexaenoic acid (DHA: C22:6n-3) in their fetuses (5.23 vs. 4.04 +/- 0.078 mg/g) compared with gilts fed the control diet. Both the flax and PFA diets increased (P < 0.05) DHA (0.60, 0.82, and 0.85 +/- 0.078 mg/g for the control, flax, and PFA diet, respectively) in the chorioallantois. In the endometrium, EPA and docosapentaenoic acid (C22:5n-3) were increased by the flax diet (P < 0.001; P < 0.05), whereas gilts receiving PFA had increased DHA (P < 0.001). The flax diet selectively increased EPA, and the PFA diet selectively increased DHA in the fetus and endometrium. In Exp. 2, gilts were fed diets containing PFA (1.5%) or a control diet beginning at approximately 170 of age (n = 13/treatment). A blood sample was collected after 30 d of treatment, and gilts were artificially inseminated when they were approximately 205 d old. Conceptus and endometrial samples were collected on d 11 to 19 of pregnancy. Plasma samples indicated that PFA increased (P < 0.005) circulating concentrations of EPA and DHA. Endometrial EPA was increased (P < 0.001) for gilts fed the PFA diet. In extraembryonic tissues, PFA more than doubled (P < 0.001) the EPA (0.13 vs. 0.32 +/- 0.013 mg/g) and DHA (0.39 vs. 0.85 +/- 0.05 mg/g). In embryonic tissue on d 19, DHA was increased (P < 0.05) by PFA (0.20 vs. 0.30 +/- 0.023 mg/g). Supplementing n-3 PUFA, beginning 30 d before breeding, affected endometrial, conceptus, and fetal fatty acid composition in early pregnancy. Dynamic day effects in fatty acid composition indicate this may be a critical period for maternal fatty acid resources to affect conceptus development and survival.

Culture and long-distance shipment of swine embryos 1☆

Abstract Embryo culture techniques were used to maintain the viability of swine embryos during shipment from the United States to England. Embryos recovered surgically from Chester White and Hampshire sows and gilts were shipped to England, then transferred to Large White gilts. Estrus was synchronized between donors and recipients by including allyl trenbolone in the daily feed for 18 and 17 consecutive days, respectively. Daily dose of allyl trenbolone was 15 mg for recipients and either 15 or 20 mg for donors. Donors were mated or artifically inseminated one to three times during estrus. During shipment, embryos were cultured in groups of six to ten in polystyrene tubes (12 × 75 mm) containing 2 ml of a modified Krebs-Ringer bicarbonate medium. The gas phase was 90% nitrogen, 5% oxygen, and 5% carbon dioxide. Culture temperature was maintained at 35 C with a temperature-controlled box, and shipment was by commerical airline. Embryos were inserted into recipient uteri 20.5 to 27 hr after recovery. In total, 227 eggs were transferred to 12 recipients, resulting in the birth of seven litters totaling 58 pigs.

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.

Synthesis of prostaglandins by pig blastocysts cultured in medium containing estradiol or catechol estrogen

Two experiments were conducted to determine the effects of 2-hydroxy-estradiol-17 beta (2-OH-E2; 0, 50 and 100 microM) and estradiol-17 beta (E2; 0, 25 and 50 microM) on prostaglandin (PG) E and PGF2 alpha synthesis by day-10 pig blastocysts (day 0 is first day of estrus). Blastocysts were incubated in a modified Krebs-Ringer bicarbonate medium, supplemented with bovine serum albumin (4 mg/ml) and the vitamins and amino acids (essential and nonessential) in Minimum Essential Medium (without phenol red or antibiotics). The incubations were conducted at 39 degrees C for three 2-h periods; the second and third periods included an E2 or catechol estrogen treatment. Release of PGF2 alpha into the culture medium decreased (p less than 0.001) linearly with increasing concentrations of 2-OH-E2 in both periods. Release of PGE was not affected by 2-OH-E2, therefore 2-OH-E2 increased (p less than 0.06) the PGE:PGF2 alpha. When E2 was added to the medium, release of PGE was decreased (p less than 0.01) during the second and third periods. Release of PGF2 alpha also was decreased (p less than 0.05) by E2 during period 2, but E2 did not alter the PGE:PGF2 alpha. Content of PGs in blastocysts at recovery was less than 10% of the PGs released in vitro. Therefore, these studies demonstrate effects of both the primary and catechol forms of E2 on the synthesis of PGE and PGF2 alpha. Catechol estrogens and E2 may inhibit PG synthesis and modify the PGE:PGF2 alpha during the establishment of pregnancy in pigs.

DEVELOPMENT OF PIG BLASTOCYSTS IN VITRO IS ALTERED BY SERUM, BOVINE SERUM ALBUMIN AND AMINO ACIDS AND VITAMINS

We tested the effects of the amino acids and vitamins in minimum essential medium (MEM) and Eagles medium (BME) on pig blastocyst development and nuclei number. Embryos were recovered either 5 or 6 d after first detected estrus and were cultured for 96 h in U-bottomed wells (0.2 ml). In Experiment 1, addition of MEM amino acids and vitamins to modified Krebs-Ringer bicarbonate (MKRB) medium containing either bovine serum albumin (BSA, 4 mg/ml) or lamb serum (10%, v/v) resulted in fewer (P<0.001) nuclei and smaller (P<0.05) embryo volumes at the end of culture as compared to embryos cultured in MKRB without MEM-supplements. Addition of MEM-amino acids without glutamine (Experiment II) depressed blastocyst volume and rate of hatching, but glutamine (2 mM) had no effect on embryo development. Dialysis (molecular weight > 12,000 retained) of fetal bovine serum (Experiment III) did not affect blastocyst expansion but reduced (P<0.05) the number of nuclei/blastocyst at the end of the culture. Embryos cultured in MKRB with dialyzed serum and the amino acids and vitamins in BME were smaller (P<0.05) and had fewer (P<0.05) nuclei than embryos cultured in MKRB with dialyzed serum but without the BME-supplements. We conclude that, under our culture conditions, MEM and BME amino acids and vitamins are detrimental to the development of early pig blastocysts and that this effect is not due to glutamine. Also, dialysis of fetal bovine serum removes some component(s) that are important for cell division by pig embryos, but it does not affect blastocyst expansion.

Non-random tissue distribution of human naïve umbilical cord matrix stem cells

AIM To determine the tissue and temporal distribution of human umbilical cord matrix stem (hUCMS) cells in severe combined immunodeficiency (SCID) mice. METHODS For studying the localization of hUCMS cells, tritiated thymidine-labeled hUCMS cells were injected in SCID mice and tissue distribution was quantitatively determined using a liquid scintillation counter at days 1, 3, 7 and 14. Furthermore, an immunofluorescence detection technique was employed in which anti-human mitochondrial antibody was used to identify hUCMS cells in mouse tissues. In order to visualize the distribution of transplanted hUCMS cells in H&E stained tissue sections, India Black ink 4415 was used to label the hUCMS cells. RESULTS When tritiated thymidine-labeled hUCMS cells were injected systemically (iv) in female SCID mice, the lung was the major site of accumulation at 24 h after transplantation. With time, the cells migrated to other tissues, and on day three, the spleen, stomach, and small and large intestines were the major accumulation sites. On day seven, a relatively large amount of radioactivity was detected in the adrenal gland, uterus, spleen, lung, and digestive tract. In addition, labeled cells had crossed the blood brain barrier by day 1. CONCLUSION These results indicate that peripherally injected hUCMS cells distribute quantitatively in a tissue-specific manner throughout the body.

Effects of L-carnitine in the Gestating Sow Diet on Fetal Muscle Development and Carcass Characteristics of the Offspring

Abstract Musser, R.E., Dritz, S.S., Davis, D.L., Tokach, M.D., Nelssen, J.L., Goodband, R.D. and Owen, K.Q. 2007. Effects of L-carnitine in the gestating sow diet on fetal muscle development and carcass characteristics of the offspring. J. Appl. Anim. Res., 31: 105–111. A total of 232 sows were used to determine the effects none or an additional 50 ppm of L-carnitine in the gestation diet on sow and offspring performance. Sows were fed dietary treatments from breeding until d 110 of gestation. Offspring from sows were ear-notched according to maternal treatment at birth and carcass data were obtained at slaughter. Sow plasma IGF-I concentration was similar (P>0.10) between control and L-carnitine supplemented sows on d 30 and 60 of gestation at 2 h after feeding. No differences were observed in either the immediate or subsequent number of pigs born per litter. No differences were observed in a sub-sample (n = 181) of pig weight at birth, weaning or d 59 of age between control and treatment offspring. Analysis of newborn pigs revealed no differences in semitendinosus cross-sectional area or primary (slow-twitch, red) fibers. Offspring of sows fed L-carnitine had a numerical tendency (P = 0.11) for a lower ratio of secondary:primary fibers compared with offspring of control sows. No differences were observed in hot carcass weight (87.6 kg); however, loin depth (57.0 < vs 59.4 mm; P < 0.01) and percentage lean (54.45 vs 55.10; P < 0.01) were increased, and backfat decreased (18.4 vs 17.8 mm: P < 0.01) in offspring of sows fed L-carnitine during gestation. Therefore, while feeding L-carnitine during gestation had no effect on the number of pigs born, it improved carcass leanness of the offspring at market weight and tended to lower ratio of secondary.—primary fibers at birth.