The stem cell continuum: a new model of stem cell regulation.

Most models of hematopoiesis have been hierarchical in nature. This is based on a large volume of correlative data. Recent work has indicated that, at least at the stem/progenitor level, hematopoiesis may, in fact, be a continuum of transcriptional opportunity. The most primitive hematopoietic stem cells are either continually cycling at a slow rate or entering and exiting cell cycle. Associated with this cycle passage are changes in functional phenotype including reversible alterations in engraftment, adhesion protein expression, cytokine receptor expression, homing to marrow, and progenitor cell numbers. Global gene expression, as measured in one point in cycle, is also markedly altered. The differentiation potential of the marrow as it transits cell cycle in response to a set differentiation stimulus also shows marked variations. This cycle-related plasticity has been clearly established for hematopoiesis. It also holds for the ability of murine marrow stem cells to home to lung and to convert to pulmonary cells. These data indicate that bone marrow stem cells can probably not be defined as discrete entities but must rather be studied on a population basis. They also indicate that mathematical modeling will become progressively more important in this field.

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.

Tolerance induction by costimulator blockade in 100 cGy treated hosts with varying degrees of genetic disparity.

Long-term multilineage allochimerism can be obtained in H2-mismatched B6.SJL to BALB/c transplants with host irradiation of 100 cGy, donor spleen cell pre-exposure and costimulator blockade with anti-CD40 ligand (CD40L) antibody. We evaluated this allochimerism approach in murine marrow transplants with different degrees of major histocompatibility complexe (MHC) mismatching; these include: (1) H2-mismatched transplant H2Kk to H2Kb, (2) full haplo-identical transplant H2Kbd to H2Kbk, (3) a partial haplo-identical transplant H2Kd to H2Kbd and (4) an MHC class II mismatch. Levels of chimerism increased up to 12 weeks and then stayed relatively stable up to 1 year after transplant. At 18 weeks post-transplant, the H2-mismatched, haplo-identical, partial haplo-identical and class II-mismatch transplants evidenced 17.9+/-4.4, 40.7+/-0.9, 25.1+/-4.19 and 33.7+/-3.5% donor chimerism, respectively. Dropping the anti-CD40 antibody treatment and spleen cells or changing the schedule of antibody to one injection, in haplo-identical or full-mismatched transplants resulted in no donor-derived chimerism. On the other hand, these still resulted in minor chimerism in class II-mismatched transplants. Lineage analysis of peripheral blood at 6 and 12 months post-transplant demonstrated a significant shift toward increased chimeric lymphocytes and decreased chimeric granulocytes in the full H2 as compared with haplo-identical or class II transplants. Transplantation with anti-CD40L antibody eliminated both graft-versus-leukemia and graft-versus-host disease (GVHD) and delayed lymphocyte infusion did not rescue animals from fatal leukemia. In conclusion, under the conditions of our tolerization regimen, a haplo transplant gives higher engraftment levels than a full H2 mismatch, and despite lower engraftment levels, a class II-mismatched transplant can be successfully accomplished with only 100 cGy and no CD40L blockade.

The marrow stem cell: the continuum.

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.

Stem cell engraftment strategies.

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 10(8) 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.

Quick sex determination of mouse fetuses.

We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in < 1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 microl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took < 4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue.

Triple MEL100 therapy in multiple myeloma.

BACKGROUND: Tandem high-dose melphalan therapy with autologous peripheral stem cell support has emerged as the standard of care for patients without prohibitive comorbidities. Mucositis and gastrointestinal side effects are the most common extrahematologic side effects. Two previously published studies presented a triple transplant with a conditioning regimen of melphalan 100 mg/m(2) (MEL100) with peripheral stem cell support every 2 to 5 months for patients with prohibitive comorbidities for high-dose tandem transplantation. We present a novel approach that investigates the triple melphalan 100/m(2) approach on a dose-dense, every-3-weeks schedule in a patient population without significant comorbidities. PATIENTS AND METHODS: Thirteen standard or high-risk patients with stage III multiple myeloma were prospectively treated. This population contained eight patients with immunoglobin G clonality, three immunoglobin A, one nonsecretory, and one light chain isotype. The induction regimens of the 13 patients were heterogenous and included five VAD, three DCIE, two Thal/Dex, two CIE, and one pulse decadron. Patients underwent peripheral blood leukopheresis, and these cells were divided into three equal sets and frozen. The patients were scheduled to receive melphalan at 100 mg/m(2) on days 1, 20, and 41, and then the autologous infusions occurred at days 0, 21, and 42. RESULTS: All patients were able to receive all three cycles of the MEL100 regimen. Seven patients (54%) received the treatments on the every-3-weeks schedule; three treatments (23%) during the second cycle and six treatments (46%) of the third cycle had to be delayed a median of 6 and 4 days, respectively. Three patients were managed completely in the outpatient setting, and the average total hospital stay for the three transplants was 18 days. Median progression-free survival was 854 days (range 73 to 1571), and the overall survival of this cohort has yet to be reached. No patient had worse than grade II mucositis, and no serious adverse events were recorded. CONCLUSION: Our regimen of three consecutive autologous peripheral stem cell transplants with a reduced dose of melphalan at 100 mg/m(2) given every 3 weeks was very well tolerated. The progression-free survival and overall survival are similar and can be compared favorably with the standard tandem myeloma regimens. Our data is intriguing, and further studies with larger numbers need to be performed to confirm these results.

Tissue injury in marrow transdifferentiation.

Recent findings indicate that adult BM contains cells that can differentiate into mature, nonhematopoietic cells of multiple tissues including cells of the kidney, lung, liver, skin and GI tract and fibers of heart and skeletal muscle. Recently the number of these observations has substantially increased, but there is a lack of information on the mechanistic issues in stem cell plasticity. In three different models for skin, liver and skeletal muscle plasticity, we have shown that following transplantation of the marrow cells from green fluorescent protein (GFP) transgenic mice, high levels of conversion of marrow cells can be identified. Injury to the tissue was the single most important factor for this phenomenon since the incidence of marrow to other tissue conversions significantly increased after tissue injury was implemented. Our studies also demonstrate the effect of radiation on the extent of marrow conversion.

Host marrow stem cell potential and engraftability at varying times after low-dose whole-body irradiation.

High levels of chimerism in syngeneic BALB/c transplants were reported when hosts were exposed to 1 Gy (100 cGy) whole body irradiation (WBI) and infused with 40 x 10(6) marrow cells. The recovery of host stem cells and alterations of enhanced host engraftability at varying times after 1 Gy WBI have now been evaluated in this study. Male BALB/c marrow (40 x 10(6) cells) was infused into female BALB/c hosts immediately or at 6, 12, and 24 weeks after 1 Gy WBI of host female BALB/c mice; engraftment percentages 8 weeks after cell injection at week 0, 6, 12, or 24 were 68% +/- 12%, 45% +/- 15%, 51% +/- 12%, or 20% +/- 8%, respectively. Eight-week engraftment levels in nonirradiated hosts average 7.7%. Conversely, engraftable stem cells measured at 8 weeks postengraftment in 1 Gy--exposed hosts were reduced to 8.6% +/- 3% of nonirradiated mice at time 0, 35% +/- 12% 6 weeks later, 49% +/- 10% at 3 months, and 21% +/- 7% at 6 months. Engraftment was still increased and stem cell decreased 1 year after 1 Gy. Furthermore, the primary cells transplanted into 1 Gy hosts can be serially transplanted, and the predominant effect of 1 Gy is directly on engrafting stem cells and not through accessory cells. These data show that transplantation in 1 Gy mice may be delayed until recovery of hematopoiesis, suggesting strategies in allogeneic transplantation to avoid the adverse effects of cytokine storm. The incomplete recovery of engraftable stem cells out to 12 months indicates that stem cell expansion, especially in patients previously treated with radiomimetic drugs, may not be feasible. (Blood. 2001;98:1246-1251)