The Heterogeneity of Clonally Derived Purified Murine Marrow Stem Cell Colonies.

Clonal stem cells have been regarded as a semi-gold standard for stem cell studies and in some instances stem cell studies have been disregarded if they were not confirmed on a clonal basis. Thus, studies of populations of stem cells have been criticized if there were not also a clonal component. This appears to be inherently contradictory. Intuitively, divergent hematopoietic stem cell function would confer an evolutionary advantage to organisms as a survival mechanism protecting against catastrophic differentiation, apoptosis or necrosis.

We have evaluated the heterogeneity of highly purified synchronized stem cells using single cell clonal cultures in cytokines and have used lineage^negativerhodamine^lowHoescht^low(LRH) murine marrow stem cells. As part of these studies, we evaluated the effect of cell cycle phase at the time of differentiation signaling using TPO, FLT3-ligand and steel factor for cycle initiation from resting G_0–1 state and then serially subcultured cells on a clonal basis prior to cell division with single cell deposition into 96-well plates determining differentiation and colony formation 14-days later. Time zero cells went directly into the differentiation subculture. Initially, we clonally subcultured cells in a granulocyte-macrophage cocktail consisting of steel factor (SCF), G-CSF and GM-CSF. Additional experiments looked at different cytokine levels and included a megakaryocytic cocktail consisting of SCF, GM-CSF, G-CSF, IL-3, IL-6, IL-11 and TPO. We have determined (using propidium iodine, tritiated thymidine and cell doubling experiments) that LRH cells enter S-phase in a synchronized fashion at about 18hrs, and exit S-phase at 40–42hrs; they double between 44–48hrs.

These studies showed a diversity of colony size, morphology and composition which was independent of cell cycle phase at time of differentiation subculture. We have analyzed a total of 800 colonies at the various time-points for morphology, cell type and cell number. We found that this “purified” population of LRH stem cells acted as individuals, i.e. snowflakes falling from the sky. There were no identical colonies. Growth in microtiter wells ranged from 0–864,600 cells (mean 77,450±296). Cloning efficiency was up to 62%, depending on cytokine concentration and combination. There was significant variability of colony size and number within as well as between time-points. In these studies, colonies were analyzed for the following differentiative hematopoietic cell lineages: neutrophilic (myeloblasts, promyelocytes, myelocytes, metamyelocytes, bands, segmented neutrophils), megakaryocytic, lymphocytic, plasmacytic, eosinophilic, basophilic, monocytic and erythocytic. We found colonies consisting of one to nine major cell types. An example highlighting the degree of heterogeneity seen, out of 55 colonies produced using SCF, GM-CSF, G-CSF at a particular time in culture and cytokine concentration, there were 33 colonies which were unique as to lineage composition. Altogether these studies show virtually complete heterogeneity of stem cells as characterized by clonal differentiation. The stem cell can probably not be defined on a single cell basis, but rather must be considered on a population basis.