A novel method for haplotype phasing in families after joint estimation of recombination fraction and linkage disequilibrium is developed. Results from Monte Carlo computer simulations show that the newly developed E.M. algorithm is accurate if true recombination fraction is 0 even for single families of relatively small sizes. Estimates of recombination fraction and linkage disequilibrium were 0.00 (SD 0.00) and 0.19 (SD 0.03) for simulated recombination fraction and linkage disequilibrium of 0.00 and 0.20, respectively. A genome fragmentation phasing strategy was developed and used for phasing haplotypes in a sire and 36 progeny using the 50 k Illumina BeadChip by: a) estimation of the recombination fraction and LD in consecutive SNPs using family information, b) linkage analyses between fragments, c) phasing of haplotypes in parents and progeny and in following generations. Homozygous SNPs in progeny allowed determination of paternal fragment inheritance, and deduction of SNP sequence information of haplotypes from dams. The strategy also allowed detection of genotyping errors. A total of 613 recombination events were detected after linkage analysis was carried out between fragments. Hot and cold spots were identified at the individual (sire level). SNPs for which the sire and calf were heterozygotes became informative (over 90%) after the phasing of haplotypes. Average of regions of identity between half-sibs when comparing its maternal inherited haplotypes (with at least 20 SNP) in common was 0.11 with a maximum of 0.29 and a minimum of 0.05. A Monte-Carlo simulation of BTA1 with the same linkage disequilibrium structure and genetic linkage as the cattle family yielded a 99.98 and 99.94% of correct phases for informative SNPs in sire and calves, respectively.

A growing number of studies conducted on diverse taxa have shown that extra-pair/group paternity is higher than what would be predicted from behavioral observations alone. While it may be beneficial for females to mate with multiple males, this often results in offspring not sired by the behavioral father, which could influence offspring survival, especially in social mammals. Feral horses (Equus caballus) maintain stable social relationships over several years, usually with one stallion defending a harem band of unrelated mares against other males. Sneak copulations by subordinate males have been observed and mares sometimes change bands, both of which can result in foals sired by males other than the dominant band stallion. We measured female fidelity in free-ranging feral horses in 23 bands, with 51 foals over four foaling seasons and tested offspring paternity against parental behaviors. We used 12 polymorphic microsatellite loci and the program CERVUS 2.0 to determine and exclude potential sires. The majority of mares remained in the band with the sire of their foal resulting in most foals being sired by the band stallion. Most foals that were not sired by the band stallion were born in the year after a round-up and we could not determine if they were the result of band changing or sneak copulations. Foals born into a band without their sire had lower survival rates and mothers were significantly more protective of foals not sired by the band stallion. These findings suggest that band stability increases the reproductive success of mares and support the importance of infanticide risk in equid social structure.

OBJECTIVE:CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS:Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS:All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION:Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes.

Objective. To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero.Materials and Methods. A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow.Results. Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well.Conclusion. We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas. Published by Elsevier Inc. on behalf of the Society for Hematology and Stem Cells (C) 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Abstract The therapeutic application of in utero hematopoietic stem cell (HSC) transplantation (IUHSCT) is theoretically attractive for definitive treatment of congenital disease states. Investigating this technique in sheep, we have previously shown long-term engraftment and expression of both allogeneic and xenogeneic donor cells without cytoablation and, under appropriate conditions, without GVHD. The theoretical basis for IUHSCT is the well-recognized immune receptivity of the fetus to engraftment of donor cells. Engraftment and long-term expression of donor human and allogeneic sheep HSC reliably occur in the fetal sheep model if the IUHSCT is performed prior to day 71 of gestation (term: 145 days), during the period of presumed immuno-naïveté. Investigations using alternate animals have also noted that gestational age at transplantation is critical to achieving long-term engraftment, presumably as a result of inducing durable immune tolerance to the donor. Despite this presumption, however, the biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. In the present studies, we investigated the role fetal immune ontogeny plays in the induction of tolerance following IUHSCT in sheep. To this end, we performed parallel experiments examining engraftment receptivity of fetal sheep to allogeneic and xenogeneic HSC and the appearance of immune phenotypes in fetal sheep lymphoid organs at varying gestational ages (days 39 to birth), attempting to draw correlations between the appearance/absence of specific immune cells and the ability to achieve durable engraftment and immune tolerance. Engraftment receptivity was determined 60 days post-transplantation at different time points in sheep fetal gestation, while immune phenotypes were determined by flow cytometry using commercially available antibodies to immune cell surface markers. Our results indicate that the fetus is largely non-receptive to engraftment of both allogeneic and xenogeneic donor HSC prior to day 52 gestation and possesses a peak in engraftment receptivity between days 64–71 of gestation, which rapidly declines thereafter. With respect to the developing fetal immune system, the period of peak engraftment receptivity was associated with the expression of CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 of gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells did not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms predominate during this time. In a similar fashion, surface IgM (sIgM)+ cells in the thymus were the first to express CD45, commencing expression around day 45 of gestation, with a comparable delay in the appearance of IgM+/CD45+ cells in the peripheral blood and spleen until late in the engraftment window. These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity/long-term engraftment and expression following IUHSCT is due to gestational age-dependent deletional tolerance. Further, our findings suggest that IUHSCT in humans may be more successful if performed during the comparable period in human gestation.

Objective: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. Methods: Engraftmentof allogeneic and xenogeneicHSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. Results: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. Conclusions: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Abstract Hemophilia A, or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 male live births. Animal models in dog, mouse, and rabbit have been developed and used to study FVIII function and to evaluate new methods of treatment and prevention of inhibitor formation. Unfortunately, for unknown reasons, results obtained using these models didn’t always result in successful therapies when applied to humans. For new treatments to be safely and successfully translated from surrogate models to clinical trials, it is critical to develop an animal model that simultaneously and accurately parallels normal human physiology while mimicking human hemophilia’s physiopathological process. Due to its striking physiological and anatomical similarities to humans, sheep are considered an ideal model to study a vast array of pathologies. The aim of these studies was to re-establish, study, and characterize an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulated human hemophilia A (ThrombHaemost68:618,1992). Thus, we used frozen semen from an affected male to generate hemophilia A carriers. We obtained 20 females that when compared to pooled control sheep plasma, exhibited slightly increased PTT levels (38.1±1.1; N=30s), normal PT and platelet number, and slightly decreased FVIII:C (70±3%). Levels of Fibrinogen, FIX, vWF activity and vWF:ag were also normal. A second round of reproductive manipulations using the carriers’ oocytes and the affected semen produced 23 more animals, 16 of which were obligate carriers with a similar phenotype. The other 8 animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII concentrate using recommended dosing. Due to the unfeasibility of clamping the umbilical cord, therapy with human FVIII was continued each 12 hours until the umbilical cord dropped off. Blood collected prior to the administration of FVIII showed that these animals had almost non-existent levels of FVIIIc, and an extremely prolonged PTT (91.5±2.9, N=30.3) with normal levels of platelets, fibrinogen, FVII, FIX, and vWF. 2 of the animals died shortly after birth due to extensive hematomas related to lambing trauma. The other 6 animals, now 5 months old (maturity 6–9 months), developed clinical symptomatology closely mimicking that of human patients with severe hemophilia A. Each of these animals had between 2–6 episodes of severe bleeding including hemarthroses of the elbow, shoulder, hip, and knee, multiple muscle hematomas, including 1 hematoma of the tongue and 1 episode of mild hematuria. All of the bleeding episodes resolved upon administration of 1–2 treatments with human FVIII. Animals have thus far received between 964-4546U of human FVIII. Of interest is that low-titer inhibitors (1.3; 2.6; 3 BU) were detected in 3 of the animals showing that the nature of the mutation present in these sheep renders them prone to inhibitor development. Characterization of the mutation is currently underway. We hope that this large animal model will contribute to a better understanding of hemophilia and the development of novel treatments that can directly translate to human patients, such as stem cell transplantation and gene therapy-based approaches.

Objective. To investigate whether the sheep xenograft model of human hematopoiesis can be used to mimic mobilization of human hematopoietic stem cells in vivo.Material and Methods. Sheep transplanted with 3.6 x 10(6) CD34(+) from human adult bone marrow were mobilized 1.5 years posttransplantation with human granulocyte colony-stimulating factor for 5 days. At day 3 and 4 of mobilization, human cells were harvested from peripheral blood (PB) and bone marrow (BM) and were injected into secondary sheep recipients (n = 6) and these animals were analyzed for the presence of human cells in their BM and PB, starting at 3.5 months posttransplantation.Results. Maximum mobilization of human cells in PB occurred at day 3, with a 21-fold increase in total numbers of human cells, and a recovery of 5.5 x 10(4)/mL CD34(+). In the BM, maximal numbers of human cells were achieved at day 4, with a 6.3-fold increase and a recovery of 1.5 x 10(4)/mL CD34(+) cells. PB and BM mobilized human cells were then transplanted into new sheep recipients, and analysis at 3.5 months posttransplantation demonstrated that levels of human cell engraftment in BM of the group transplanted with mobilized PB were significantly lower than those transplanted with BM cells (0.6% +/- 0.1% vs 8.0% +/- 1.8%). Furthermore, in sheep transplanted with mobilized PB, the levels of human cells in circulation remained 2.5-fold higher than the levels of human cells found in their BM.Conclusion. Mobilization of human cells in the sheep model parallels human PB and BM hematopoietic stem cells (HSC) mobilization in healthy human donors in their ability to engraft, differentiate, and repopulate secondary hosts. Thus, this model can become a useful tool to study mobilization regimens, mechanisms, and quality of products obtained. (c) 2007 ISEH-Society for Hematology and Stem Cells. Published by Elsevier Inc.

Transplantation of hematopoietic stem cells into fetal sheep early in gestation (day (d) 55) (term gestation = 145d) results in long-term multi-lineage hemopoietic chimerism. We have proposed that the fetus is immunologically receptive at this time. However, our understanding of the mechanisms underlying this establishment of fetal tolerance remains poorly defined. We hypothesized that examination of developmental events in fetal immune ontogeny might provide insights into mechanisms underlying fetal tolerance formation. To this end we have previously reported the early appearance of cells phenotypically consistent with T regulatory cells (CD4+/CD25+) and multilineage expression of the CD45 isoform CD45R early in gestation (d40–65). Here, we extend our observations on B lineage cell distribution and relative CD45 isoform expression during gestation. The two commercially-available ovine CD45 antibodies are CD45 (immunoprecipitates proteins of MW 225, 210, and 190 kD, (Immunology. 1985; 55:347–353)) and CD45R (immunoprecipitates a single protein of MW 220 kD (Cell. Immunol. 1987; 110:46–55)). Using dual-color flow cytometry, cells from fetal thymus, intestine, lung, spleen, bone/bone marrow, and peripheral blood (PB) were analyzed for expression of surface IgM, CD45 and CD45R from d39 to two days after birth. Surface IgM+ cells in the thymus were observed to be as high as 28% at d39 of gestation (uniformly CD45R+/CD45−). This rapidly dissipated to less than 1% after d65 of gestation, although IgM+ cells were observed as late as two days after birth. All IgM+ cells expressed CD45R in all organs tested but relative CD45 expression varied by gestational age and organ. Subsequent sampling noted rapid onset of CD45 coexpression on >70% of detectable thymic surface IgM+ cells throughout gestation. Similarly, splenic IgM+ cells co-expressed high levels of CD45 while PB IgM+ cells had delayed CD45 expression (beyond d65). Bone/bone marrow showed variable coexpression of CD45 throughout gestation. By contrast, surface IgM+ cells in lung and intestine maintained CD45R expression without coexpression of CD45 prior to birth. We have previously reported humoral tolerance to retroviral vector transgene products transplanted at d55–60 (Blood. 2001; 97:3417–3423). Further understanding of CD45 isoform expression patterns and relative organ distribution of surface IgM+ cell populations during the time of immune receptivity in the sheep may provide insight into fetal transplantation tolerance.