Christina McAuliffe

Marrow Stem Cells Shift Gene Expression and Engraftment Phenotype with Cell Cycle Transit

We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3′ end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from “stem cell genes” being highly expressed at 0 h and turned off at 48 h, while “cell division” genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.

Homing of Purified Murine Lymphohematopoietic Stem Cells: A Cytokine-Induced Defect

This study was designed to establish a direct homing assay using purified lineage-negative Sca-1-positive (Lin(-) Sca(+)) murine bone marrow cells and to evaluate the effects of cytokines on homing. C57BL/6 Lin(-) Sca(+) marrow stem cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and then injected by tail vein into untreated C57BL/6 mice. Marrow was harvested at various times after cell infusion and analyzed on a high-speed MoFlo cell sorter for fluorescent positive events, using a large event analysis, with at least 16 million total events analyzed. We have shown that homing of Lin(-) Sca(+) cells plateaus by 1 h, and at 3 h post-infusion is linear between 50,000 and 1,000,000 infused cells. This forms a base for a homing assay in which 250,000 CFDA-SE labeled Lin(-) Sca(+) marrow cells are infused and then recovered from marrow 3 h later, followed by a large-event fluorescence-activated cell sorting (FACS) analysis. We found that 7.45-9.32% of infused cells homed and that homing of stem cells cultured for 48 h in interleukin-3 (IL-3), IL-6, IL-11, and steel factor cultured cells was defective when compared to noncultured cells. Exposure of marrow stem cells to IL-3, IL-6, IL-11, and steel factor induces a stem cell homing defect, which probably underlies the engraftment defect previously characterized under these conditions.

The fleet feet of haematopoietic stem cells: rapid motility, interaction and proteopodia

Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high‐speed optical‐sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage‐negative, rhodamine‐low, Hoechst‐low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three‐dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal‐derived factor‐1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.

Cytokine receptor repertoire and cytokine responsiveness of Hodull/Rhdull stem cells with differing potentials for G1/S phase progression

OBJECTIVE Subsetting of Hoechst 33342 dull (Ho(dull)) hematopoietic stem cells on the basis of rhodamine 123 (Rh) efflux utilizing an improved dual-dye efflux strategy resolves Ho(dull)/Rh(dull) stem cell subsets that differ with regard to their rate of recruitment and progression through the cell cycle upon exposure to cytokines. MATERIALS AND METHODS Murine bone marrow cells were isolated by negative immunomagnetic selection using lineage-directed antibodies followed by Ho and Rh staining using a dual-dye efflux method. RESULTS Ho(dull)/Rh(dull) stem cells that efflux Rh more efficiently (R1) exhibit a 4- to 8-hour delay in progression to S phase when stimulated by interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) compared to Ho(dull)/Rh(medium) stem cells, which retain low levels of Rh (R2). R1 and R2 cells show a hierarchical entry into S phase upon exposure to any or all of these cytokines. The R1 subset contains proportionately more high proliferative potential colony-forming cells than the R2 subset, but equivalent levels of engraftable stem cells at 3 and 8 weeks after competitive transplantation. Both R1 and R2 cells express c-kit, IL-3R, and IL-11R, whereas IL-6R and c-fms are only expressed by R1 or R2 cells, respectively. Cytokine stimulation of R1 and R2 cells induced cell cycle progression with elevated or induced expression of c-kit, c-fms, IL-2R, and IL-6R. CONCLUSION These studies indicate that primitive marrow stem cells can be further subsetted by degree of Rh staining to reveal important functional phenotypic differences between cells with different levels of Rh staining.

A method for clonal analysis of epidermal growth factor-responsive neural progenitors

Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10-20 ng/ml EGF for 12-14 days. This gave a cloning efficiency of 2.12%+/-0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10-14 and 21-28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells.

Circadian variations of bone marrow engraftability

Circadian rhythms exist for hematopoiesis, but little is known about circadian variation of bone marrow engraftability and host “acceptability”. Using a B6.SJL to C57BL/6J congenic transplant model, we chose 3‐times with light on: Hours After Light Onset (HALO) 4, 8, and 12 and 3‐times with light off: HALO 16, 20, and 24. The mice were conditioned on a 12‐h light/dark cycles. Recipient mice (100 cGy) received 40 million cells. We demonstrated a significant variation of bone marrow engraftability into bone marrow, spleen, and thymus when donor animals were subjected to changes in their light/dark cycles. Two statistically significant nadirs in all three organs were observed at HALO 8 and 24 in experiments carried out in July, while an identical set of experiments in February analyzing engraftment in marrow and spleen showed nadirs at HALO 8, but not at HALO 24. Marrow progenitors from the July experiments showed nadirs at HALO 12 and 24. The percentage of progenitors in S phase peaked at HALO 8 and 24. Interestingly, there were no changes in the ability of host to accept grafts with changes in the light/dark cycles of host animals. Circadian variations of bone marrow engraftability are important and should be considered in bone marrow transplant strategies.

RHYTHMICITY OF ENGRAFTMENT AND ALTERED CELL CYCLE KINETICS OF CYTOKINE-CULTURED MURINE MARROW IN SIMULATED MICROGRAVITY COMPARED WITH STATIC CULTURES

SummarySpace flight with associated microgravity is complicated by “astronauts anemia” and other hematologic abnormalities. Altered erythroid differentiation, red cell survival, plasma volume, and progenitor numbers have been reported. We studied the impact of microgravity on engraftable stem cells, culturing marrow cells in rotary wall vessel (RWV) culture chambers mimicking microgravity and in normal gravity nonadherent Teflon bottles. A quantitative competitive engraftment technique was assessed under both conditions in lethally irradiated hosts. We assessed 8-wk engraftable stem cells over a period spanning at least one cell cycle for cytokine (FLT-3 ligand, thrombopoietin [TPO], steel factor)-activated marrow stem cells. Engraftable stem cells were supported out to 56 h under microgravity conditions, and this support was superior to that seen in normal-gravity Teflon bottle cultures out to 40 h, with Teflon bottle culture support superior to RWV from 40 to 56 h. A nadir of stem cell number was seen at 40 h in Teflon and 48 h in RWV, suggesting altered marrow stem cell cycle kinetics under microgravity. This is the first study of engraftable stem cells under microgravity conditions, and the differences between microgravity and normal gravity cultures may present opportunities for unique future stem cell expansion strategies.

The marrow stem cell: the continuum.

Summary:The marrow hematopoietic stem cell is currently being redefined as to all aspects of its phenotype and its total differentiation capacity. This redefinition now includes its plasticity as to production of nonhematopoietic and hematopoietic cell types, the determinants of its in vivo engraftment potential and its expression of stem cell functional characteristics.

Lymphohematopoietic stem cell engraftment

Abstract: Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step; in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation.

Marrow stem cell potential within a continuum.

Abstract: On the basis of our studies of the fluctuation of the hematopoietic stem cell phenotype with cell cycle trnsit, we hypothesize that the ability of marrow stem cells to convert to nonhematopoietic cells will also vary at different points in the cell cycle. The new biology of stem cells has an impact on many fields including developmental biology and stem cell biology and the clinical potential is enormous.