Jane E. Carlson

Murine marrow cellularity and the concept of stem cell competition: geographic and quantitative determinants in stem cell biology

In unperturbed mice, the marrow cell numbers correlate with the stem cell numbers. High levels of long-term marrow engraftment are obtained with infusion of high levels of marrow cells in untreated mice. To address the issue of stem cell competition vs ‘opening space’, knowledge of total murine marrow cellularity and distribution of stem and progenitor cells are necessary. We determined these parameters in different mouse strains. Total cellularity in BALB/c mice was 530±20 million cells; stable from 8 weeks to 1 year of age. C57BL/6J mice had 466±48 million marrow cells. Using these data, theoretical models of infused marrow (40 million cells) replacing or adding to host marrow give chimerism values of 7.5 and 7.0%, respectively; the observed 8-week engraftment of 40 million male BALB/c marrow cells into female hosts (72 mice) gave a value of 6.91±0.4%. This indicates that syngeneic engraftment is determined by stem cell competition. Our studies demonstrate that most marrow cells, progenitors and engraftable stem cells are in the spine. There was increased concentration of progenitors in the spine. Total marrow harvest for stem cell purification and other experimental purposes was both mouse and cost efficient with over a four-fold decrease in animal use and a financial saving.

Overcoming Fas-Mediated Apoptosis Accelerates Helicobacter-Induced Gastric Cancer in Mice

The initiating molecular events in Helicobacter-induced gastric carcinogenesis are not known. Early in infection, Fas antigen-mediated apoptosis depletes parietal and chief cell populations, leading to architectural distortion. As infection progresses, metaplastic and dysplastic glands appear, which are resistant to Fas-mediated apoptosis. These abnormal lineages precede, and are thought to be the precursor lesions of, gastric cancer. Acquisition of an antiapoptotic phenotype before transformation of cells suggests that loss of Fas sensitivity may be an early required trait for gastric cancer. We reasoned that forced Fas-apoptosis resistance would result in earlier and more aggressive gastric cancer in our mouse model. Fas antigen-deficient (lpr) mice or C57BL/6 wild-type mice were irradiated and reconstituted with C57BL/6 marrow forming partial lpr/wt chimera or wt/wt control mice, extending the life span of the lpr and ensuring a competent immune response to Helicobacter felis infection. Infected lpr/wt mice developed gastric cancer as early as 7 months after infection (compared with 15 months in wt/wt mice). At 10 months (90%) and 15 months (100%), mice developed aggressive invasive lesions. This earlier onset and more aggressive histology strongly argues that Fas-apoptosis resistance is an early and important feature of gastric cancer formation.

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.

Stem Cell Engraftment Strategies

Abstract: The donor stem cell phenotype and host microenvironment determine the outcome of a stem cell transplant. In a series of transplant studies in syngeneic male to female or congenic Ly5.1/Ly5.2 models in which hosts have received no or minimal irradiation (100 cGy), evidence overwhelmingly supports the concept that syngeneic engraftment is determined by stem cell competition. These approaches can be extended to H‐2 mismatched allogeneic mouse combination when antigen pre‐exposure and CD40‐CD40 ligand antibody blockage are employed. A human trial in patients with resistant neoplasia infusing pheresed blood with 108 CD3 cells/kg showed that tumor responses and complete chimerism occur with very low levels of CD34+ cells/kg and that the extent of previous treatment is a critical factor in determining chimerism. A major feature of transplants is the phenotype of the donor stem cell. This phenotype shows dramatic reversible plasticity involving differentiation, adhesion protein expression, and engraftment with cytokine‐induced cell‐cycle transit. Homing is probably also plastic. Marked fluctuations in engraftment capacity are also seen at different points in marrow circadian rhythm.

Major histocompatibility complex class II inhibits fas antigen-mediated gastric mucosal cell apoptosis through actin-dependent inhibition of receptor aggregation.

ABSTRACT Escape from normal apoptotic controls is thought to be essential for the development of cancer. During Helicobacter pylori infection, the leading cause of gastric cancer, activation of the Fas antigen (Fas Ag) apoptotic pathway is responsible for early atrophy and tissue loss. As disease progresses, metaplastic and dysplastic glands arise which express Fas Ag but are resistant to apoptosis and are believed to be the precursor cells for adenocarcinoma. In this report, we show that one mechanism of acquired Fas resistance is inhibition of receptor aggregation via a major histocompatibility complex class II (MHCII)-mediated, actin-dependent mechanism. For these studies we used the well-described C57BL/6 mouse model of Helicobacter pylori and Helicobacter felis infection. Under normal conditions, Fas Ag is expressed at low levels, and MHCII expression on gastric mucosal cells is negligible. With infection and inflammation, both receptors are upregulated, and 6.1% of gastric mucosal cells express MHCII in combination with Fas Ag. Using the rat gastric mucosal cell line RGM-1 transfected with murine Fas Ag and MHCIIαβ chains, we demonstrate that MHCII prevents Fas receptor aggregation and inhibits Fas-mediated signaling through its effects on the actin cytoskeleton. Depolymerization of actin with cytochalasin D allows receptors to aggregate and restores Fas sensitivity. These findings offer one mechanism by which gastric mucosal cells acquire Fas resistance.

Bone marrow cells as the origin of stomach cancer.

Cells derived from bone marrow are pluripotent, with the ability to differentiate into multiple cell types. Environmental cues dictate differentiation decisions. It should not be surprising then, that abnormal cell environments lead to abnormal differentiation of these cells, and in some cases, malignant transformation. Identifying a role for bone marrow-derived cells in the initiation and progression of cancer allows a dramatic change in the way in which cancer is viewed. Identifying the cell responsible for initiating a tumor offers the exciting possibility of specifically targeting unique aspects of these cells and altering signaling properties for more effective therapeutic approaches.

Evaluation of mouse whole body bone marrow cellularity and distribution of hematopoietic progenitors

Abstract In order to perform murine transplant experiments, marrow cells are usually harvested from hind legs bones either by bone marrow flushing or bone crushing. We examined the feasibility of harvesting whole skeleton marrow in regards to cell numbers and progenitor potential. Bones of BALB/c mice were dissected and flushed with cold HBSS or crushed in a mortar with cold HBSS buffer. The obtained solution was filtered on a 40 μm strainer, then washed once. Cells were counted with a hemocytometer. For HPP analysis, 20,000 cells were plated in soft agar with 7 factors. Total marrow cell counts averaged 525 ± 34 × 10 6 cells/mouse (n = 7). Comparison of flushed hind legs (2 tibias, femurs, iliac) with crushed hind legs gave 114 and 96 × 10 6 cells, respectively. Crushed spine gave 170 × 10 6 cells. The progenitor content of each region, compared by HPP, is shown in the table (colonies/20,000 plated). Significant increase of progenitors was found between flushed and crushed legs and between legs and spine. Despite showing more total colonies, whole body showed equal percentages of HPP compared to crushed legs. The difference observed between flushed and crushed legs may indicate a cortical localization of progenitors. Total marrow harvest is feasible, economical and provides high numbers of hematopoietic cells rich in progenitors.

Influence of timing of administration of 5-fluorouracil to donors on bone marrow engraftment in nonmyeloablated hosts

We evaluated the engraftment and the cell cycle status of marrow cells at various times after 5-fluorouracil (5-FU) administration. 5-FU (150 mg/kg) was given to donor male BALB/c mice at 1, 2, 6, or 12 days prior to marrow harvest. The donor cells were then assessed in host nonmyeloablated female mice. Bone marrow engraftment of marrow treated with 5-FU was evaluated and compared to marrow treated with diluent (phosphate-buffered saline) at 3 and 10 weeks after marrow infusion. Our data show a rapid induction of an engraftment defect 1 day after 5-FU, persistence of this defect through day 6, and a recovery by day 12. Experiments using hydroxyurea (which selectively kills cells in the S phase) to determine the cell cycle status indicated that cells that engrafted in post-5-FU marrow were noncycling at days 1, 2, and 12 but cycling at day 6. Post-5-FU bone marrow was also analyzed in vitro by colony assays and its cycling status determined by3H-thymidine suicide assay. High-proliferative-potential colony-forming cells (HPP-CFCs) and low-proliferative-potential colony-forming cells (LPP-CFCs) decreased rapidly 1 day after 5-FU, with a nadir observed at day 6 for HPP-CFCs and day 2 for LPP-CFCs. By day 12, LPP-CFCs showed a total recovery, but HPP-CFCs were still defective. Significant numbers of HPP-CFCs were cycling, mostly at days 6 and 8 after 5-FU, whereas LPP-CFCs appeared quiescent except at day 2. These results emphasize the importance of timing if post-5-FU marrow is used for gene therapy or marrow transplantation.

Definitive evidence for the influence of circadian rhythm on bone marrow (Bm) Stem cell engraftability

Abstract Numerous studies have demonstrated the influence of circadian rhythm on the hematopoietic system but little is known about variation of BM engraftability throughout the day. We transplanted 40 × 10 6 Ly5.1 (B6.SJL) BM cells into submyeloablated (100 cGY) Ly5.2 (C57BL/6J) recipients. Donors were kept isolated in 2 custom built cabinets in order to ensure 12 hours of light and 12 hours of dark. Recipients were also synchronized in order to receive the transplant consistently at Circadian Time (CT) 8 (2 pm). We had 15 donors and 15 recipients for each CT. For transplant, marrow was pooled into three groups of five. Percent (± SD) engraftment was determined by the ratio Ly5.2/(Ly5.1 + Ly5.2). There was a significant fluctuation (p These in vivo observations were complemented by in vitro analysis of donor BM. Colony-assays (HPP-CFC), thymidine suicide tests (percentage of cycling cells), and lineage analysis were performed. Significant asynchronous fluctuations of HPP-CFC numbers were seen while proliferation varied inversely with engraftment. These data provide statistically significant evidence of circadian variations of BM engraftability and form a base for further studies of stem cell chronobiology.