Functional and molecular characterization of a primitive bone marrow -derived stem cell

One of the challenges facing investigators working in the field of hematopoietic stem cell (HSC) biology has been to develop a strategy to purify this rare cell type to homogeneity. Our laboratory has established a unique protocol to isolate mouse HSCs on the basis of the ability of these cells to home to bone marrow. This functional assay obtains a primitive cell (2-day homed cell) that exhibits both hematopoietic and epithelial engraftment capabilities. An essential component of this protocol is the collection of a small sized cell by counter-flow elutriation. Due to the limited accessibility of this technique we sought to provide the scientific community with an alternate approach to acquire these primitive stem cells. Here we describe a means to combine density gradient separation technology with the 2-day homing protocol. We show that high density bone marrow cells can be used to obtain cells capable of providing long-term hematopoiesis for lethally irradiated recipients at limiting dilutions. Little is known about the molecular characteristics of the 2-day homed cell. We performed microarray analysis to examine the gene expression profile of these primitive cells and to provide molecular evidence to support their developmental plasticity. We established the molecular signature of the 2-day homed cell and determined that it differs significantly from a HSC isolated by cell surface phenotyping exhibiting limited plasticity. Using a Molecular Signatures Database, the 2-day homed cell was found to share a number of genes with mouse embryonic and neural stem cells. We identified common genes that may be potential targets for future studies of plasticity. We also evaluated the use of an in vitro assay to assess the developmental potential of the 2-day homed cell. This assay utilized spontaneous embryoid body formation to generate chimeras consisting of mouse embryonic stem cells and 2-day homed cells. Only low numbers of 2-day homed cells were able to integrate into the developing embryoid body and few cells were capable of survival. Thus, these adult marrow-derived stem cells apparently retain multipotency, share genes with other primitive stem cell types and do not survive in the milieu associated with embryoid body formation.