R. Michael Roberts

Derivation of induced pluripotent stem cells from pig somatic cells

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after ≈22 days, providing an overall reprogramming efficiency of ≈0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of ≈17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.

Pregnancy-Associated Bovine and Ovine Glycoproteins Exhibit Spatially and Temporally Distinct Expression Patterns During Pregnancy

Abstract The pregnancy-associated glycoproteins (PAG) constitute a large family of recently duplicated genes. They show structural resemblance to pepsin and related aspartic proteinases. A total of 21 bovine (bo) PAG and 9 ovine (ov) PAG cDNA have been identified. Phylogenetic analysis indicated that the PAG are divided into two main groupings that accurately reflect their tissue expression, as determined by in situ hybridization. In the first pattern, represented by ovPAG-2 and boPAG-2, -8, -10, and -11 (where the numbering is arbitrary and reflects order of discovery within species), expression occurred throughout the outer epithelial layer of the placenta (trophectoderm). The second pattern was predominant localization to binucleate cells. Ribonuclease protection assays, which allow discrimination between closely related transcripts, have shown that the expression of PAG varies in a temporal manner over pregnancy. Of those bovine PAG expressed predominantly in binucleate cells, boPAG-1, -6, and -7 are expressed weakly, if at all, by Day 25 placenta, but are present at the middle and end of pregnancy. Others, such as boPAG-4, -5, and -9, are expressed at Day 25 and at earlier stages. Although not among the earliest PAG produced by the trophoblast, boPAG-1 has been used for pregnancy diagnosis, particularly in dairy cows, where there is a major need for a sensitive method capable of detecting pregnancy within 1 mo of conception. It seems likely that some of the newly discovered PAG will be better candidates than PAG-1 for pregnancy diagnosis.

Maternal Diet and Other Factors Affecting Offspring Sex Ratio: A Review

Abstract Mammals usually produce approximately equal numbers of sons and daughters, but there are exceptions to this general rule, as has been observed in ruminant ungulate species, where the sex-allocation hypothesis of Trivers and Willard has provided a rational evolutionary underpinning to adaptive changes in sex ratio. Here, we review circumstances whereby ruminants and other mammalian species, especially rodents and primates, appear able to skew the sex ratio of their offspring. We also discuss some of the factors, both nutritional and nonnutritional, that potentially promote such skewing. Work from our laboratory, performed on mice, suggests that age of the mother and maternal diet, rather than the maternal body condition per se, play directive roles in controlling sex ratio. In particular, a diet high in saturated fats but low in carbohydrate leads to the birth of significantly more male than female offspring in mature laboratory mice, whereas when calories are supplied mainly in the form of carbohydrate rather than fat, daughters predominate. As the diets fed to the mice in these experiments were nutritionally complete and because litter sizes did not differ between treatments, dietary inadequacy seems not to be the cause for sex-ratio distortion. A number of mechanisms, all of which are testable, are discussed to provide an explanation for the phenomenon. We conclude the review by discussing potential implications of these observations to human medicine and agriculture.

Sexual dimorphism among bovine embryos in their ability to make the transition to expanded blastocyst and in the expression of the signaling molecule IFN-τ

IFN-τ is a secretory product of trophectoderm of cattle, sheep, and their relatives and is expressed for a few days in early pregnancy after the blastocyst first forms. It serves to alert the mother that she is pregnant. A delayed or less than robust IFN-τ signal is a likely cause of embryonic loss. Here we have determined whether blastocyst production of IFN-τ, which is encoded by a cluster of genes on chromosome 9, differs between the sexes in cattle, as assessed by culture of in vitro-derived embryos on two different media, one complex (tissue culture medium 199 supplemented with serum) with coculture support, the other relatively simple (synthetic oviductal fluid plus albumin). With both media, female blastocysts produced approximately double the amount of IFN-τ as males, regardless of such variables as oocyte batch, blastocyst quality, hatching, and length of time in culture. However, in either tissue culture medium 199, which contains 5.5 mM d-glucose, or in synthetic oviductal fluid, in the presence but not in the absence of added glucose, significantly fewer female than male embryos were able to progress from the morula/early blastocyst stage to more advanced stages of development. It is possible that the differences between male and female embryos both in their production of IFN-τ and in their ability to progress in development in glucose-rich media are manifestations of phenomena that occur in vivo and provide plasticity in embryo selection during early pregnancy.

Differentiation of Induced Pluripotent Stem Cells of Swine into Rod Photoreceptors and Their Integration into the Retina

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real‐time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment‐specific membrane protein 1 in outer segment‐like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation. STEM CELLS 2011;29:972–980

Striking variation in the sex ratio of pups born to mice according to whether maternal diet is high in fat or carbohydrate

In female mammals, it remains controversial whether maternal diet and particularly the source and availability of energy can influence sex of offspring born. Outbred female mice were fed ad libitum from 30 days to ≈45 wk of age on defined, complete diets that differed only in their relative content of fat and carbohydrate to determine whether calorie source influenced litter size and sex ratio of pups. Diet 1 (very high in saturated fat, VHF) provided 60% of calories as fat, mainly lard. Diet 2 (low in saturated fat, LF) was low in fat (10% of calories) but high in carbohydrate. Mice delivered four litters of pups, resulting in a total of 1,048 young born over 108 pregnancies. Gestation length and litter size did not differ between VHF and LF groups and did not change as mice aged. Sex ratio of pups (fraction male) born to mothers on VHF diet was unusually high (0.67) and to mothers on LF diet very low (0.39) over litters 2, 3, and 4. This skewing of sex ratio was related to diets fed and not to body mass of mothers. Age of mothers was an important variable, however. Mice that were first bred at 10 wk of age delivered similar numbers of sons and daughters, whereas virgin mice bred later than 20 wk of age produced litters that were skewed toward males or females according to diet. The data show that the source of calories provided in a nutritionally complete diet to mature female mice can influence sex of offspring born.

Trophoblast Stem Cells

Trophoblast stem cells (TSC) are the precursors of the differentiated cells of the placenta. In the mouse, TSC can be derived from outgrowths of either blastocyst polar trophectoderm (TE) or extraembryonic ectoderm (ExE), which originates from polar TE after implantation. The mouse TSC niche appears to be located within the ExE adjacent to the epiblast, on which it depends for essential growth factors, but whether this cellular architecture is the same in other species remains to be determined. Mouse TSC self-renewal can be sustained by culture on mitotically inactivated feeder cells, which provide one or more factors related to the NODAL pathway, and a medium supplemented with FGF4, heparin, and fetal bovine serum. Repression of the gene network that maintains pluripotency and emergence of the transcription factor pathways that specify a trophoblast (TR) fate enables TSC derivation in vitro and placental formation in vivo. Disrupting the pluripotent network of embryonic stem cells (ESC) causes them to default to a TR ground state. Pluripotent cells that have acquired sublethal chromosomal alterations may be sequestered into TR for similar reasons. The transition from ESC to TSC, which appears to be unidirectional, reveals important aspects of initial fate decisions in mice. TSC have yet to be derived from domestic species in which remarkable TR growth precedes embryogenesis. Recent derivation of TSC from blastocysts of the rhesus monkey suggests that isolation of the human equivalents may be possible and will reveal the extent to which mechanisms uncovered by using animal models are true in our own species.

Identification of Oxygen-Sensitive Transcriptional Programs in Human Embryonic Stem Cells

To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O(2) atmosphere (approximately 4% O(2)), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O(2) conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O(2). Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O(2). Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O(2), while a few genes implicated in lineage specification were up-regulated. Although the differences between O(2) conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O(2) favors the molecular mechanisms required for the maintenance of pluripotency.

Relationship between age of blastocyst formation and interferon-τ secretion by in vitro–derived bovine embryos

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon‐τ production. A total of 800 oocytes were fertilized by frozen‐thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two‐, four‐, and eight‐cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50‐μl droplets of medium, fixed for cell counts, and the concentration of interferon‐τ in the medium was determined. Blastocysts originating from two‐cell embryos had significantly fewer cells (46.5 ± 23.3) than either four‐cell‐ (97.2 ± 13.5) or eight‐cell‐derived embryos (113.8 ± 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 ± 15.5 versus 41.9 ± 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight‐ or four‐cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two‐cell embryos (941.7 ± 92.1, 930.1 ± 163.1, versus 232.8 ± 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon‐τ secretion. The embryos age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon‐τ production, with mean concentrations in the medium of 294.8 ± 57.9, 563.3 ± 82.0, 1126.3 ± 133.6, 1778.5 ± 297.2, 512.9 ± 82.0, 315.0 ± 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon‐τ than those that form on days 9 or 10. Since early‐forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon‐τ production and competence. Mol. Reprod. Dev. 49:254–260, 1998.

Caprine pregnancy‐associated glycoproteins (PAG): Their cloning, expression, and evolutionary relationship to other PAG

Pregnancy‐associated glycoproteins (PAG) are structurally related to aspartic proteinases and belong to an extensive, rapidly evolving family of recently duplicated genes expressed in the placentas of artiodactyl species. The aim of the present study was to clone PAG from the goat, study their temporal and cell‐specific expression, and determine their phylogenetic relationship to PAG from other species. RT‐PCR was used to generate PAG cDNA from pooled placental RNA obtained between days 45 and 115 of pregnancy. A total of 11 cDNA, which differed by > 5% from each other, were selected for complete bidirectional sequencing from 60 clones analyzed. A group of nine (caPAG1, caPAG3–7var, caPAG9–11), which displayed > 80% sequence identity with each other, were expressed after day 45 of pregnancy and were localized to trophoblast binucleate cells. These PAG demonstrated an unusually high ratio of nonsynonymous (amino acid changing) to synonymous nucleotide differences. CaPAG2, by contrast, was detectable only in early pregnancy (days 18 and 19) and expressed throughout trophectoderm. It was of more ancient origin than the PAG1 group, but more recent than caPAG8. The latter was expressed at all stages examined (days 18 to 115). The data confirm that many PAG genes, with different patterns of temporal and spatial expression, are transcribed in the placenta of the goat. The data also suggest that the recently duplicated PAG genes are being selected for rapid diversification of function. Mol. Reprod. Dev. 57:311–322, 2000.