Stem Cells and Development

Health-related quality of life (QOL) is poorest during the immediate post-transplantation period, but the impact of medical interventions during this period has not been studied. Colony-stimulating factors(CSFs), which are used to minimize short-term negative outcomes, might be expected to improve QOL; however, little is published about their impact on QOL during this period. We conducted a MEDLINE search to identify studies reporting on outcomes of stem cell transplantation (SCT) affected by the CSFs, mainly sargramostim and filgrastim. End points studied were: mucositis, incidence and type of infection, duration of hospitalization, time to myeloid engraftment, and quantity and quality of harvested cells. To impute the impact of CSFs on QOL post-SCT, we also reviewed the association between QOL and CSF outcomes in other circumstances. Data suggest that both CSFs improve QOL in the early autologous or allogeneic post-bone marrow transplantation period. Poor QOL caused by infection and increased length of hospital stay is expected to be improved by sargramostim. Time to myeloid engraftment, when negatively affecting QOL, is expected to be improved with both CSFs; however, the time to myeloid engraftment is consistently shorter with filgrastim. Current prospective trials designed to study the effects of CSFs in the immediate post-SCT period should collect QOL data.

Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Administration of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) results in mobilization of PBSCs into the peripheral blood. G-CSF and GM-CSF differ somewhat in the number and composition of CD34⁺ cells and effector cells mobilized to the peripheral blood; however, the molecular mechanism underlying the release and engraftment of CD34⁺ cells by these growth factors is poorly understood. This review provides a recent update on the involvement of hematopoietic growth factors, chemokines, adhesion molecules, and chemokine receptors in the regulation of stem cell release and engraftment. The involvement of very late antigen-4 (VLA-4), VLA-5, leukocyte function associated-1 molecule (LFA-1), and L-selectin and their receptors CXCR4 and its ligand SDF-1 will be discussed, and cross talk between these factors will also be reviewed in the context of stem cell release and engraftment. Finally, PBSC mobilization by chemokines will be reviewed in relation to hematopoietic growth factors.

We generated a monoclonal antibody (mAb) 6G7, which recognizes a 220-kD antigen on selected subpopulations of normal myeloid and lymphoid cells and their malignant counterparts. 6G7 reacts with 90-95% of peripheral blood B cells, 70-80% of CD8⁺ cells, 30-35% of CD4⁺ cells, 20-40% of monocytes, and 20-40% of CD34⁺ cells from bone marrow. 6G7 reacts with leukemic blasts in acute myeloid leukemia (14/16), adult acute lymphoblastic leukemia (ALL) (5/5), pediatric ALL (5/9), chronic lymphocytic leukemia (8/8), follicular lymphoma (7/7), and Burkitt's lymphoma (1/1). Long-term bone marrow culture of 6G7^+/- cells showed the majority of clonogenic hematopoietic cells were in mAb 6G7 subpopulation. An immunotoxin of 6G7 and ricin A chain was cytotoxic to 6G7⁺ leukemia cell lines. mAb 6G7 has potential clinical applications for targeted immunotherapy of both leukemia and lymphoma.

Relapse after adjuvant chemotherapy or high-dose chemotherapy with stem cell transplant for high-risk breast cancer remains high and new strategies that provide additional antitumor effects are needed. This report describes methods to generate highly effective HER2/neu-specific cytotoxic T cells by arming activated T cells with anti-CD3 × anti-HER2/neu bispecific antibody (BsAb). OKT3 and 9184 (anti-HER2) monoclonal antibodies (mAb) were conjugated and used to arm T cells that were subsequently tested in binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregated and specifically killed HER2/neu⁺ breast cancer cells. Cytotoxicity emerged after 6 days of culture, was higher in armed T cells than unarmed T cells at all effector to target ratios (E/T) tested, and increased as the arming dose was increased. At an E/T of 20:1, the mean cytotoxicity of armed activated T cells (ATC) from 10 normal subjects increased by 59 ± 11% (±SD) over that seen in unarmed ATC (p< 0.001) and the mean cytotoxicity of armed ATC from 6 cancer patients increased by 32 ± 9% above that seen for unarmed ATC (p < 0.0004). After arming, the BsAb persisted on ATC up to 72 h and armed ATC continued to be cytotoxic up to 54 h. The amount of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte-macrophage colony-stimulating factor(GM-CSF) secreted was 1699, 922, and 3092 pg/ml/10⁶ cells per 24 h, respectively, when armed T cells were exposed to a HER2/neu⁺ breast carcinoma cell line. These studies show the feasibility and clinical adaptability of this approach for generating large numbers of anti-HER2-specific, cytotoxic T cells for clinical trials.

On the horizon of modern molecular medicine is the requisite technology to capture multipotent human stem cells that are capable of self-renewal and to direct these stem cells along defined lineages for therapeutic purposes. In this article, we describe the hematopoietic and mesenchymal differentiation potential of a unique population of transforming growth factor-β1 (TGF-β1)-responsive stem cells derived from murine bone marrow. Stringent selection of the stem cells was accomplished under low serum conditions by virtue of an inherent survival response to a TGF-β1-vWF fusion protein that was bound to collagen matrices. The TGF-β1-responsive stem cells initially exhibited a non-adherent and uniformly blastoid morphology, underwent expansion into colonies upon serum reconstitution, and were capable of overt cytodifferentiation along fibrogenic, osteogenic, chondrogenic, or adipogenic lineages upon growth factor stimulation. Remarkably, these stem cells also underwent rapid expansion in the presence of either hematopoietic stem cell factor (SCF) or interleukin3 (IL-3), and differentiated into myeloid and lymphoid phenotypes upon exposure to the latter. Taken together, these results support the hypothesis that pluripotent premesenchymal prehematopoietic progenitor cells, designated P4 stem cells, are present postnatally in murine bone marrow and, thus, may be summarily isolated for various cell-based experimental therapies.

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133⁺ and CD34⁺ cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133⁺ and CD34⁺ fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133⁺ CB cells was up to 1262-fold for total cells, 99-fold for CD34⁺ cells, 109-fold for CD34⁺ CD133⁺ cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19⁺), NK (CD56⁺), and T (CD4⁺ CD8⁺) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133⁺ or CD34⁺ cells. Thus, the CD133⁺ subset could be expanded in the same manner as the CD34⁺ subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.

Ex vivo activation of peripheral blood stem cells (PBSC) using interleukin-2 (IL-2) results in cytotoxic effector cells that may possess beneficial in vivo effects. We proposed to evaluate ex vivo stimulation of PBSC using various cytokines alone or in combination to optimize their function. Cytokine-activated PBSC were analyzed for tumor-directed cytotoxicity and their ability to remove tumor cells from long-term clonogenic assays. Mononuclear cells were obtained from the apheresis products of normal donors and cultured with IL-2 (1000 U/ml), interferon-α (IFN-α) (1000 U/ml), or IL-12 (50 U/ml) either alone or in combinations at 37°C and 5% CO₂ for 24 h. Colony-forming unit-tumor (CFUT) assays were initiated using cytokine-activated PBSC with varying concentrations of MCF-7 or SKBR-3 human breast cancer cells. Standard 4-h ⁵¹Cr-release assays were performed with cytokineactivated PBSC using MCF-7 or SKBR-3 cells as targets. Activation of PBSC with IL-2, IFN-α , or IL-12 resulted in enhanced cytotoxicity against the two breast cancer cell lines when compared to controls. PBSC activated with IL-2 and IFN-α or IL-2 and IL-12 were more cytotoxic than PBSC activated with single cytokines (p = 0.0004 for MCF-7 cells and p < 0.001 for SKBR-3 cells). Using clonogenic assays, IL-2-activated PBSC reduced the number of CFU-T to a greater extent than did IL-12 or IFN-α-activated PBSC (p = 0.0006). However, PBSC activated with a combination of IL-2 and IFN-α or IL-2 and IL-12 demonstrated 95% and 90% reductions, respectively, compared to 79% reduction using IL-2-activated PBSC (p < 0.0001). The greatest reduction in cytotoxicity occurred in the cell populations depleted of CD56⁺ cells (p = 0.016) and CD8⁺ CD56⁺ cells (p= 0.002), suggesting that the effector cell population includes a combination of cytotoxic CD8⁺ T cells and CD56⁺ natural killer cells. These results demonstrate that the ex vivo activation of PBSC with cytokines, either alone or in combination, enhances cytotoxicity against, and removal of two human breast cancer cells. The combinations of IL-2 with IFN-α or IL-12 are most beneficial in cytotoxicity and purging assays. These results could play an important role in designing adoptive cellular immunotherapy clinical trials in the autologous hematopoietic stem cell transplant setting.

We have studied the adhesion of human marrow CD34⁺ precursors to stromal layer of nontransformed human marrow myofibroblasts (normal stroma) and to different stromal cell lines immortalized by T (PU-34) or t and T (L88/5, L87/4, L2Ori-, KM-102) oncogenes from simian virus 40 and E6, E7 oncogenes from human papilloma virus 16 (HS-27A, HS-23). Flow cytometry and Western blotting studies showed that cells from all lines were stromal myofibroblasts similar to normal stroma. Using an original method of adhesion measurement, we found that adhesion of CD34⁺ cells was significantly increased on PU-34 cell layer as compared to normal stroma (43% vs. 27%) whereas adhesion on HS-27A and HS-23 was significantly decreased (11% and 8.5%, respectively), and adhesion on L88/5, L87/4, KM-102 and L2Ori- was negligible to nil (<6%). Adhesion of CD34⁺ cells to stromal layers paralleled the expression of α smooth muscle (αSM) actin within the microfilaments of the cells from the different lines and was inversely correlated to their anchorage-independent growth in semisolid agar. These data show that adhesion to the stromal layer of CD34⁺ cells is related to the αSM actin microfilamentous network in marrow myofibroblasts and that transformation can negatively affect this microfilamentous network and therefore adhesion of hematopoietic precursors.

Tumor cell contamination of stem cell collections harvested from breast cancer patients is a common phenomenon described by several investigators but with findings that vary among reports. Although so-called co-mobilization of these cells has been hypothesized, the origin of tumor cell contamination in stem cells is still unknown. A total of 47 G-CSF mobilized stem cell grafts from patients with nodal-positive(n = 30), chemosensitive metastatic (n = 11), and 5 women with inflammatory breast cancer were evaluated for cancer cells by immunocytochemistry. Additionally, 40 bone marrow aspirations and 23 peripheral blood samples collected prior to apheresis and after one to two cycles of conventional chemotherapy were available for examination. Tumor cell contamination of leukapheresis correlated best with preharvest blood state. This was valid when the nominal (positive/negative) presence of tumor cells in blood was compared to the nominal presence of tumor cells in apheresis samples and when the it was correlated to the tumor cell load of apheresis samples (TCL = tumor cells per 10⁶ nucleated cells investigated). The correlation between blood and stem cells was better (nominal and quantitative) than that between marrow and stem cells, despite the larger sample size of marrow aspirations. The presence or absence of cancer cells in apheresis samples could not be safely predicted by the presence or absence of tumor cells in marrow or blood alone. Diagnostic specificity seems to improve from a combination of results from marrow and blood analysis. No correlation was found in quantitative analysis of tumor cell contamination between marrow and blood. In conclusion, the results suggest that blood and bone marrow represent different compartments for epithelial cancer cells and that contaminating tumor cells in stem cell harvests may be derived from the blood and/or marrow compartment. The tumor cell contamination of a stem cell harvest cannot be safely predicted by a preceding blood or marrow analysis.

Umbilical cord blood as an alternative source of hematopoietic stem cells and nonmyeloablative conditioning are very exciting new developments in transplantation. We report here mixed chimerism in two adult patients with severe aplastic anaemia using nonmyeloablative conditioning and two-antigen mismatched cord blood transplantation resulting in satisfactory clinical response. Our results suggest that such transplant is possible with minimal toxicity.

Seventeen patients with refractory follicular lymphoma heavily treated with chemotherapy (>2 regimens), radiotherapy, and biological modifiers were enrolled in a pilot study to receive six weekly doses, instead of the more frequent four doses, of monoclonal anti CD20, at a standard dose of 375 mg/m². In an intent-to-treat analysis, overall response was 76%, of which 47% (8 patients) were a complete response. With a median follow-up of 33.6 months, 7 complete responders remained alive and free of disease, and 2 partial-response patients remained stable without additional treatment. Actuarial curves showed that at 3 years, 53% of patients should be alive and free of disease. The 4 patients who were failures died secondary to tumor progression. Overall survival at 3 years was 76%. Toxicity was mild; all patients completed the schedule on time and doses. The addition of two doses of anti-CD 20 clearly improved the outcome in a group of patients with refractory follicular lymphoma heavily treated and poor prognostic factors. However, the number is too small to drawn definitive conclusions, and more clinical trials are necessary to determine if four of six doses of anti-CD20 therapy are better in this setting of patients.

The effect of granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood progenitor cells (PBPC) on the endogenous serum levels of cytokines stem cell factor (SCF) and flt3-ligand(flt3-L) was studied in 18 healthy subjects undergoing allogeneic PBPC donation. Donors received a standardized mobilization regime consisting of a 4-day course of G-CSF, with leukapheresis on day 5. Endogenous serum flt3-L and SCF were determined prior to G-CSF administration, on the day of leukapheresis, and followed up until day +100 after cessation of G-CSF administration. The administration of G-CSF resulted in a transient elevation of endogenous flt3-L serum levels. At the day of leukapheresis serum flt3-L showed a median increase of 75% compared to serum flt3-L levels obtained before G-CSF treatment. The increase in serum flt3-L levels showed no correlation with the total number of progenitor cells mobilized. Cessation of G-CSF treatment led to normalization of serum flt3-L within 7 days post G-CSF administration. In contrast, serum CSF levels remained unchanged in response to G-CSF administration. Our results demonstrate a transient surge in serum flt3-L in relation to G-CSF-induced PBPC mobilization, although the assessment of endogenous flt3-L give no information regarding the ability for G-CSF-induced PBPC recruitment in healthy individuals.