Sally E. Johnson

Equine umbilical cord blood contains a population of stem cells that express Oct4 and differentiate into mesodermal and endodermal cell types.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem‐like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast‐like morphology and express the stem cell markers Oct4, SSEA‐1, Tra1‐60 and Tra1‐81. Culture of the cells in tissue‐specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic differentiation reveals proteoglycan and glycosaminoglycan synthesis as measured histochemically and Sox9 and collagen 2A1 gene transcription. Osteocytes capable of mineral deposition, osteonectin and Runx2 transcription were evident. Hepatogenic cells formed from UCBs express albumin and cytokeratin 18. Multinucleated myofibers that express desmin were observed indicating partial differentiation into mature muscle cells. Interestingly, conventional human protocols for UCB differentiation into adipocytes were unsuccessful in foal UCB and adult horse adipose‐derived MSC. These results demonstrate that equine UCB can be induced to form multiple cell types that underlie their value for regenerative medicine in injured horses. In addition, this work suggests that subtle differences exist between equine and human UCB stem cells. J. Cell. Physiol. 215: 329–336, 2008.

Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis. However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified. We examined the role of Raf-mediated signaling as a modulator of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype. The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis. Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

Fibroblast Growth Factor 2 Promotes Primitive Endoderm Development in Bovine Blastocyst Outgrowths

Primitive endoderm (PE) is the second extraembryonic tissue to form during embryogenesis in mammals. The PE develops from pluripotent cells of the blastocyst inner cell mass. Experimental results described herein provide evidence that FGF2 stimulates PE development during bovine blastocyst development in vitro. Bovine blastocysts were cultured individually on a feeder layer-free, Matrigel-coated surface in the presence or absence of FGF2. A majority of blastocysts cultures formed outgrowths (76.8%) and the rate of outgrowth formation was not affected by FGF2 supplementation. However, supplementation with FGF2 increased the incidence of PE outgrowths on Days 13 and 15 after in vitro fertilization. Presumptive PE cultures contained cells with a phenotype distinct from trophectoderm (TE). Cell identity was validated by expression of GATA4 and GATA6 mRNA and transferrin protein, all markers of the PE lineage. Expression of GATA4 occurred coincident with blastocyst expansion and hatching. These cells did not express IFNT and CDX2 (TE lineage markers). Profiles of FGF receptor (FGFR) isoforms were distinct between PE and TE cultures. Specifically, FGFR1b and FGFR1c were the predominant FGFR transcripts in PE whereas FGFR2b transcripts were abundant in TE. Supplementation with FGF2 increased the mitotic index of PE but not TE. Moreover, FGF signaling appears important for initiation of PE formation in blastocysts, presumably by lineage committal from NANOG-positive epiblast cells, because chemical disruption of FGFR kinase activity with PD173074 reduces GATA4 expression and increases NANOG expression. Collectively, these results indicate that FGF2 and potentially other FGFs specify PE formation and mediate PE proliferation during early pregnancy in cattle.

Insulin-like growth factor I stimulates myoblast expansion and myofiber development in the limb

Insulin‐like growth factor I (IGF‐I) is expressed in the anterior and posterior mesodermal cells of the developing limb. However, a definite role for IGF‐I during early limb organogenesis is unknown. To determine the inherent participation of IGF‐I during limb organ development, a retroviral delivery system (RCAS) was used to overexpress IGF‐I throughout the developing hind limb of stage 24 chicken embryos. The area of the belly of the external gastrocnemius muscle in the IGF‐I infected limb was an average of 160, 90, 70, and 80% larger than the contralateral control muscle belly, 4, 5, 6, and 7 days postinjection, respectively (all differences P < 0.01). In comparison to the contralateral control muscles, there were a significantly greater number of muscle fibers in the IGF‐I infected muscles (P < 0.05), confirming that the majority of IGF‐I–mediated muscle enlargement was due to an increase in total fiber numbers (hyperplasia). Four days postinjection, there was a 32% increase in myoblast to myofiber ratio in the muscle of injected limbs compared with the muscle in the contralateral noninjected control limbs (P < 0.05). This result demonstrates that IGF‐I acts to expand the undifferentiated myoblast population, and as a result, more myofibers subsequently develop, and the muscles expressing ectopic IGF‐I are enlarged by means of hyperplasia. There was no difference in tibiotarsus and fibula length or diameter between the IGF‐I injected and control limb, suggesting that ectopic IGF‐I expression within the mesoderm was not a nonspecific growth stimulant of all tissues of the developing limb, but specifically enhanced skeletal muscle development and growth. Ectopic IGF‐I expression had no significant effect on myostatin mRNA concentrations. Our results support a model where mesodermally expressed IGF‐I acts to regulate the number of primary myofibers, and, therefore, size of skeletal muscles, which form during the initial events of limb myogenesis.

Thioredoxin Reductase Regulates Angiogenesis by Increasing Endothelial Cell-Derived Vascular Endothelial Growth Factor

Abstract: Low selenium (Se) status increases angiogenesis by inducing the production of vascular endothelial growth factor (VEGF); however, the mechanism responsible for VEGF up-regulation has yet to be characterized. Ses ability to control cellular oxidative state through its incorporation into selenoproteins such as thioredoxin reductase (TrxR) may explain previous studies that connect Se status to tumor angiogenesis. Therefore, the focus of this study was to determine if altered VEGF expression and angiogenesis due to decreased Se levels are influenced by reduced TrxR activity. We found that chemical inhibition of TrxR in Se-sufficient endothelial cells (ECs) was associated with increases in VEGF and VEGF receptor expression, cell migration, proliferation, and angiogenesis to levels similar to those seen in Se-deficient ECs. Specific inhibition of glutathione peroxidase did not affect pro-angiogenic responses, indicating a unique role of the TrxR system during low Se status. These data correlate changes in TrxR activity with changes in VEGF expression and angiogenic development in ECs, which is significant because minimal mechanistic data exist that explain the role of Se in cancer prevention. Understanding the importance of the tumor microenvironment in contributing to angiogenic regulation has the potential to significantly impact breast cancer chemoprevention strategies by focusing on maintaining proper EC function within the mammary gland.

Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation

Niche localized HGF plays an integral role in G(0) exit and the return to mitotic activity of adult skeletal muscle satellite cells. HGF actions are regulated by MET initiated intracellular signaling events that include recruitment of SHP2, a protein tyrosine phosphatase. The importance of SHP2 in HGF-mediated signaling was examined in myoblasts and primary cultures of satellite cells. Myoblasts stably expressing SHP2 (23A2-SHP2) demonstrate increased proliferation rates by comparison to controls or myoblasts expressing a phosphatase-deficient SHP2 (23A2-SHP2DN). By comparison to 23A2 myoblasts, treatment of 23A2-SHP2 cells with HGF does not further increase proliferation rates and 23A2-SHP2DN myoblasts are unresponsive to HGF. Importantly, the effects of SHP2 are independent of downstream ERK1/2 activity as inclusion of PD98059 does not blunt the HGF-induced proliferative response. SHP2 function was further evaluated in primary satellite cell cultures. Ectopic expression of SHP2 in satellite cells tends to decrease proliferation rates and siSHP2 causes an increase the percentage of dividing myogenic cells. Interestingly, treatment of satellite cells with high concentrations of HGF (50 ng/ml) inhibits proliferation, which can be overcome by knockdown of SHP2. From these results, we conclude that HGF signals through SHP2 in myoblasts and satellite cells to directly alter proliferation rates.

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS‐FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin‐expressing, connective‐tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked‐cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

17β-Hydroxyestra-4,9,11-trien-3-one (Trenbolone) preserves bone mineral density in skeletally mature orchiectomized rats without prostate enlargement

Testosterone enanthate (TE) administration attenuates bone loss in orchiectomized (ORX) rats. However, testosterone administration may increase risk for prostate/lower urinary tract related adverse events and polycythemia in humans. Trenbolone enanthate (TREN) is a synthetic testosterone analogue that preserves bone mineral density (BMD) and results in less prostate enlargement than testosterone in young ORX rodents. The purpose of this experiment was to determine if intramuscular TREN administration attenuates bone loss and maintains bone strength, without increasing prostate mass or hemoglobin concentrations in skeletally mature ORX rodents. Forty, 10 month old male F344/Brown Norway rats were randomized into SHAM, ORX, ORX+TE (7.0mg/week), and ORX+TREN (1.0mg/week) groups. Following surgery, animals recovered for 1 week and then received weekly: vehicle, TE, or TREN intramuscularly for 5 weeks. ORX reduced total and trabecular (t) BMD at the distal femoral metaphysis compared with SHAMs, while both TREN and TE completely prevented these reductions. TREN treatment also increased femoral neck strength by 28% compared with ORX animals (p<0.05), while TE did not alter femoral neck strength. In addition, TE nearly doubled prostate mass, compared with SHAMs (p<0.05). Conversely, TREN induced a non-significant 20% reduction in prostate mass compared with SHAMs, ultimately producing a prostate mass that was 64% below that found in ORX+TE animals (p<0.01). Hemoglobin concentrations and levator ani/bulbocavernosus (LABC) muscle mass were elevated in ORX+TE and ORX+TREN animals to a similar degree above both SHAM and ORX conditions (p<0.01). In skeletally mature rodents, both high-dose TE and low-dose TREN completely prevented the ORX-induced loss of tBMD at the distal femoral metaphysis and increased LABC mass. TREN also augmented femoral neck strength and maintained prostate mass at SHAM levels. These findings indicate that TREN may be an advantageous agent for future clinical trials evaluating agents capable of preventing bone loss resulting from androgen deficiency.

Protein Kinase C Delta Mediates Fibroblast Growth Factor-2-Induced Interferon-Tau Expression in Bovine Trophoblast

Interferon-tau (IFNT) is the trophoblast-secreted factor responsible for establishing and maintaining pregnancy in ruminants. Several uterine- and embryo-derived factors, including fibroblast growth factor-2 (FGF2), regulate IFNT production. The objective of the present study was to decipher the intracellular signaling mechanisms employed by FGF2 to regulate IFNT production. In bovine trophoblast cells (CT1), mitogen-activated protein kinase kinase-dependent pathways mediated constitutive IFNT mRNA concentrations. However, FGF2-mediated increases in IFNT mRNA levels occurs independent of mitogen-activated protein kinase. Exposure to the pan-protein kinase C (PKC) inhibitor, calphostin C, did not affect basal IFNT mRNA levels but limited the ability of FGF2 to increase IFNT mRNA abundance. Also, supplementation with phorbol 12-myristate 13-acetate (PMA) stimulated IFNT mRNA levels to the same extent as with FGF2. PMA and FGF2 cosupplementation did not elicit an additive effect on IFNT mRNA abundance. Pharmacological antagonists for classic PKCs (Gö6976) or novel PKCs, including PKC delta (rottlerin), were used to identify the specific PKC isoform utilized by FGF2. Supplementation of CT1 cells with Gö6976 did not affect FGF2 or PMA activities, whereas rottlerin prevented FGF2- and PMA-dependent increases in IFNT mRNA abundance in CT1 cells. Rottlerin also prevented FGF2 from increasing IFNT mRNA levels in Vivot trophoblast cells and primary trophoblast outgrowths. Modifications in PRKCD phosphorylation status were evident following FGF2 and PMA treatment. Also, reducing PRKCD expression by RNA interference attenuated FGF2-dependent increases in IFNT mRNA abundance. In conclusion, these results provide evidence that FGF2 regulates IFNT production in bovine trophectoderm by acting through PRKCD.

Subspecies differences in early fetal development and plasma pregnancy-associated glycoprotein concentrations in cattle

Inclusion of Bos indicus genetics improves production traits of cattle maintained in hot climates. Limited information exists detailing pregnancy-specific events as influenced by variable amounts of Bos indicus genetics. Three experiments were completed to examine the effect of Bos taurus and Bos indicus genotypes on fetal size and plasma pregnancy-associated glycoprotein (PAG) concentrations. In all experiments, cows were bred by AI after synchronization of ovulation. Fetal measurements were completed by transrectal ultrasonography and plasma PAG concentrations were quantified from plasma harvested the day of each fetal measurement. In Exp. 1, fetal size and plasma PAG concentrations were measured at d 53 of pregnancy in cows composed of various fractions of Angus and Brahman (n = 9 to 21 cows/group). Fetus size was greater in cows containing >80% Angus genetics compared with cows containing <80% Angus influence (3.40 ± 0.28 vs. 2.86 ± 0.28 cm crown-rump length; P < 0.01). Plasma PAG concentrations were reduced (P < 0.01) in cows containing >80% Angus genetics when compared with their contemporaries (6.0 ± 1.5 ng/mL vs. 9.4 ± 1.5 ng/mL). In Exp. 2, fetal measurements and plasma PAG concentrations were determined at d 35 and 62 of pregnancy in Angus and Brangus cows. Breed did not affect fetus size at d 35, but Angus cows contained larger fetuses than Brangus cows at d 62 [3.0 ± 0.03 vs. 2.8 ± 0.03 cm crown-nose length (CNL; P > 0.01)]. Plasma PAG concentrations were not different between breed at d 35 and 62 (P > 0.1). In Exp. 3, fetal measurements and plasma samples were collected at d 33/34, 40/41, 47/48, and 54/55 post-AI in Angus and Brangus cows. Fetus size was not different (P > 0.05) between genotypes on d 33/34, 40/41, and 47/48. Angus fetuses were larger than Brangus fetuses at d 54/55 (2.1 ± 0.03 vs. 1.9 ± 0.03 cm CNL; P = 0.001). Plasma PAG concentrations were less in Angus than Brangus cows at each time point (average 4.9 ± 0.9 vs. 8.2 ± 0.9 ng/mL; P = 0.005). In conclusion, these studies determined that the Bos taurus × Bos indicus genotype impacts fetal size and rate of fetal development by 7 wk of gestation. Plasma PAG concentrations were increased in cattle with Bos indicus genetics in 2 of 3 studies, suggesting that genotype is one of several determinants of PAG production and secretion in cattle.