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Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. Stem Cells 2014;32:662–673

Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.

Recombinant G-CSF has been given to over 150 normal donors for the collection of allogeneic or syngeneic peripheral blood stem cells (PBSC). G-CSF was found to be well-tolerated with mild-moderate bone pain, edema and mild thrombocytopenia being the observed side effects. To date, approximately 90 unmodified primary PBSC transplants from HLA-identical related donors have been performed with engraftment that is, in general, considerably more rapid than marrow. Acute graft-versus-host-disease (GVHD), grades II-IV occurred in 47% of patients and grades III-IV in 17%. Despite the infusion of one to two logs more T cells, these results are not remarkably different than would be expected with marrow transplantation. There have also been successful reports of using G-CSF mobilized allogeneic PBSC following second transplants for graft rejection or relapse. Allogeneic PBSC have been infused without reconditioning for correction of graft failure and unmodified or CD34 selected PBSC have also been given with marrow to augment the dose of hematopoietic cells. Further studies are needed to define the role of allogeneic PBSC for transplantation, refine PBSC mobilization and collection techniques and to evaluate the long-term effects of cytokines in normal donors.

Recombinant G-CSF has been given to over 150 normal donors for the collection of allogeneic or syngeneic peripheral blood stem cells (PBSCs). G-CSF was found to be well-tolerated with mild-moderate bone pain, edema and mild thrombocytopenia being the observed side effects. To date, approximately 100 unmodified primary PBSC transplants from HLA-identical related donors have been performed with engraftment that is, in general, considerably more rapid than marrow. Acute graft-versus-host-disease (GVHD) grades II — IV occurred in 44% of patients and grades III — IV in 16%. From a small number of evaluable patients surviving for more than 100 days, it appears the incidence of chronic GVHD is approximately 50%. Despite the infusion of one to two logs more T cells, these results are not remarkably different than would be expected with marrow transplantation. Further studies are needed to define the role of allogeneic PBSCs for transplantation, to refine PBSC mobilization and collection techniques, and to evaluate the long-term effects of cytokines in normal donors.

Using restriction analysis of polymerase chain reaction (PCR)-amplified DNA, the cytomegalovirus (CMV) envelope glycoprotein (gB and gH) genotypes were determined for virus isolates from 128 bone marrow transplant recipients with fatal or nonfatal CMV. All isolates could be assigned to one of four gB and gH genotypes previously identified by DNA sequencing studies. Isolates of gB type 1 were more commonly found to be of gH type 2, whereas gB types 2-4 were more commonly linked to gH type 1. A small frequency of recombination with gB was detected by restriction analysis of DNA from variable regions of the gp55 and gp116 domains. Multiple isolates from various sites of 29 patients were typed and, with three exceptions, the gB genotype remained constant in all isolates from a single patient. Patients who survived CMV infection more commonly shed virus of gB type 1 than those who died (P = .003). This significant difference of gB types among patient subsets is unexplained but raises the possibility that gB genotypes may serve as a marker for pathogenicity of CMV strains in marrow transplant patients.