Haplotype phasing after joint estimation of recombination and linkage disequilibrium in breeding populations.

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.

Hematologic and IgG responses of heifers experimentally infected with the agent of epizootic bovine abortion.

BACKGROUND: Epizootic bovine abortion (EBA) is a tick-transmitted abortive disease of beef cattle in the western United States. Infected cattle do not have clinical signs until abortion occurs, usually within the last trimester of gestation. There is little information on the hematologic response of the dam following infection. OBJECTIVE: The purpose of this study was to determine if changes in blood leukocytes and serum IgG concentrations could be detected following experimental infection of pregnant heifers with the etiologic agent of EBA (aoEBA). METHODS: Twelve Angus heifers were infected during gestation with the aoEBA using an inoculum prepared from the thymus of an infected fetus. Five pregnant heifer controls were given an inoculum prepared from the thymus of an aoEBA-negative calf. PCVs, total and differential leukocyte counts, and serum IgG concentrations were measured weekly following administration of the inocula until abortion or calving. Gross and microscopic examinations were performed on all aborted fetuses to confirm infection. RESULTS: Eleven of 12 heifers in the treatment group aborted, and significant findings were decreased lymphocyte counts at weeks 1 and 14 postinoculation and increased monocyte counts at week 4 compared with control animals. Serum IgG concentrations were significantly increased at weeks 6-8 and 11 in the treatment group. CONCLUSIONS: Leukogram changes are subtle in infected cattle. Future research efforts should be aimed at development of an antibody test specific for detection of previously infected animals, which could graze safely on EBA-endemic pastures.

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells.

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.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells.

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.

Immune ontogeny and engraftment receptivity in the sheep fetus.

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: Engraftment of allogeneic and xenogeneic HSC 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.

Development and characterization of a novel CD34 monoclonal antibody that identifies sheep hematopoietic stem/progenitor cells.

OBJECTIVE: We and many others have long used sheep as a predictive model system in which to explore stem cell transplantation. Unfortunately, while numerous markers are available to identify and isolate human hematopoietic stem cells (HSC), no reagents exist that allow HSC/progenitors from sheep to be identified or purified, greatly impeding the application of this well-established large animal model to the study of autologous or allogeneic HSC transplantation. The current studies were undertaken to create a monoclonal antibody to sheep CD34 that would enable isolation and study of sheep HSC/progenitors. MATERIALS AND METHODS: A partial cDNA to the extracellular domain of the sheep CD34 antigen was polymerase chain reaction cloned, characterized, and used to genetically immunize mice and create hybridomas. RESULTS: The resultant monoclonal antibody to sheep CD34 allows flow cytometric detection of sheep HSC/progenitors present within bone marrow, cord blood, and mobilized peripheral blood. Moreover, this antibody can be used to enrich for HSC/progenitors with enhanced in vitro colony-forming potential, and also identifies endothelial cells in situ within paraffin-embedded tissue sections, similarly to antibodies to human CD34. CONCLUSIONS: The availability of this monoclonal antibody recognizing the stem cell antigen CD34 in sheep will greatly facilitate the study of autologous and allogeneic HSC transplantation using this clinically relevant large animal model.

The human-sheep chimeras as a model for human stem cell mobilization and evaluation of hematopoietic grafts' potential.

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.