PRONOUNCED HETEROGENEITY OF CLONALLY DERIVED PURIFIED MURINE MARROW STEM CELLS

PRONOUNCED HETEROGENEITY OF CLONALLY DERIVED PURIFIED MURINE MARROW STEM CELLS

Huerta, Felix

Cell cycle position seems to be important in stem cell function and differentiation. We have previously shown reversible function and phenotypic changes in the hematopoietic stem cell as it transverses cell cycle using a primitive acting cytokine culture to synchronously drive cells through culture. We have looked at engraftment, progenitor expression, adhesion protein expression, cytokine receptor expression, gene expression and homing ability of stem cells through cycle. This has led to a continuum model of stem cell regulation and we hypothesized that the differentiative potential of primitive stem cells shifts during cycle transit. We have cultured lineage^sup negative^ rhodamine^sup low^ Hoescht^sup low^ (LRH) murine marrow stem cells with thrombopoietin, FLT3-ligand and steel factor (FTS) of cycle initiation from resting G^sub 0^ state and then serially sub-culturing cells on a clonal basis with single cell deposition into 96 well plates after primary cell cycle transit using FTS and have subcultured them in steel factor, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), determining differentiation 14 days later. Time zero cells went directly into the sub-culture of G-CSF, GM-CSF and steel factor. We have determined that LRH cells enter S-phase in a synchronized fashion at about 18 hours, left S-phase at 40-42 hours and doubled between 44-48 hours using tritiated thymidine and cell doubling experiments. Clonal studies, evaluating these cells on an individual basis gave us a totally different picture. What was highlighted was a diversity of colony size, morphology and composition. We have now analyzed a total of 150 colonies at the various time-points for morphology, cell type and cell number. We found this “purified” population of LRH stem cells acted as individuals, i.e., snowflakes falling from the sky. Not one colony was identical to each other. Colony size range from 0-8082 cells (mean 1216±268). There was significant variability of colony size and number within as well as between time points. Total cell count by time in culture, correlating with cell cycle position showed at 18 hrs (G^sub 1^-S) there was a mean of 2078±276 cells per colony, compared to 688±110 at 32hrs (S phase), 945±144 at 40hrs (late S/earlyG^sub 2^) and a mean of only 188 cells at Ohrs (G^sub 0-1^). Altogether these results highlight the heterogeneity of even a highly purified population of cells comprising in its

FELIX HUERTA, MD, MUTHALAGU RAMANATHAN, MD, IMAD BITAR, MD, MARK DOONER, MD, MEHRDAD ABEDI, MD, DELIA DEMERS, MD, DEBBIE GREER, MD, JEAN-FRANCOIS LAMBERT, MD, GERALD A. COLVIN, MD, PETER J. QUESENBERRY, MD

ROGER WILLIAMS MEDICAL CENTER, DEPARTMENT OF RESEARCH, ADEL DECOF CANCER CENTER

Copyright Rhode Island Medical Society Dec 2004

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