A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow.

BACKGROUND: Isolation of bone marrow cells, including hematopoietic stem cells, is a commonly used technique in both the research and clinical settings. A quantitative and qualitative assessment of cell populations isolated from mouse and human bone marrow was undertaken with a focus on the distribution of hematopoietic cells between the central bone marrow (cBM) and endosteal bone marrow (eBM). METHODS: Two approaches to cBM isolation from the hind legs were compared using the C57BL/6J and BALB/cJ strains of laboratory mice. The content of hematopoietic stem cells in eBM was compared to cBM from mice and human fetal bone marrow using flow cytometry. Enzymatic digestion was used to isolate eBM and its effects on antigen expression was evaluated using flow cytometry. Humanized immunodeficient mice were used to evaluate the engraftment of human precursors in the cBM and eBM and the effects of in vivo maturation on the fetal stem cell phenotype were determined. RESULTS: The two methods of mouse cBM isolation yielded similar numbers of cells from the femur, but the faster single-cut method recovered more cells from the tibia. Isolation of eBM increased the yield of mouse and human stem cells. Enzymatic digestion used to isolate eBM did, however, have a detrimental effect on detecting the expression of the human HSC-antigens CD4, CD90 and CD93, whereas CD34, CD38, CD133 and HLA-DR were unaffected. Human fetal HSCs were capable of engrafting the eBM of immunodeficient mice and their pattern of CD13, CD33 and HLA-DR expression partially changed to an adult pattern of expression about 1 year after transplantation. CONCLUSIONS: A simple, rapid and efficient method for the isolation of cBM from the femora and tibiae of mice is detailed. Harvest of tibial cBM yielded about half as many cells as from the femora, representing 6.4 % and 13 %, respectively, of the total cBM of a mouse based on our analysis and a review of the literature. HSC populations were enriched within the eBM and the yield of HSCs from the mouse and human long bones was increased notably by harvest of eBM.

A diverse group of small circular ssDNA viral genomes in human and non-human primate stools.

Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated.

The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny.

The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117(++)CD203c(+) mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38-CD34(++) and CD133(+)CD34(++) cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver.