Stem Cell Preparations Research Articles

The Placental/Umbilical Cord Blood Program of the New York Blood Center. A progress report.

The transplantation of placental/umbilical cord blood (P/CB) has been used successfully to reconstitute bone marrow function in both related and unrelated recipients. We report here the experience of the New York Blood Center P/CB Program. Since its inception in 1992, over 400 unrelated transplants were supported between July 1993 and September 1997. Overall, event-free survival for all diagnoses and ages approached 0.45. Success and rapidity of engraftment correlated most strongly with the degree of human leukocyte antigen (HLA) disparity and cell dose/kg body weight recipient. Acute graft-versus-host disease (GVHD) was common in all patients but, surprisingly, did not differ between those patients who received grafts having one or more antigen mismatches. Chronic GVHD was uncommon and only rarely contributed to death. These results demonstrate the feasibility of large-scale P/CB banking for the provision of cryopreserved stem cell preparations for unrelated transplants. The degree of the program's success argues strongly for additional P/CB banks in order to increase the likelihood of finding a suitable stem cell preparation for patients for whom related matched donors do not exist.

Sequential immunotyping and genotyping of tumor cells in bone marrow of cancer patients: a model study.

Epithelial cells can be detected in bone marrow or peripheral stem cell preparations of patients with various kinds of cancer and their presence in bone marrow is of prognostic significance. Characterization of these cells has been hampered by their low frequency. Here we present a method that may allow sequential immunophenotyping and genotyping of epithelial cells in bone marrow. To simulate in vivo situations, cells from the colon cancer cell line HT29 were seeded into bone marrow and were first detected by the Fab fragment of the A45-B/B3 anticytokeratin antibody. Expression of Ki67, p53, Her-2/ neu (c-erbB2), and 17-1A could be detected on A45-B/B3-stained cells by immunofluorescence using a fluorescein-labeled anti-mouse immunoglobulin specific for the Fc part of mouse immunoglobulins. Reactivity for all antigens except for Ki67 persisted after A45-B/B3 labeling even when a scoring step for the presence of epithelial cells was performed before proceeding with the immunophenotyping. After immunophenotyping, numerical chromosomal aberrations and amplifications of the Her-2/neu oncogene could be assessed by fluorescence in situ hybridization in the same A45-B/B3-stained cells. This combination of immunophenotyping and genotyping may help in establishing the role of epithelial cells in bone marrow or peripheral stem cell harvests for tumor relapse and formation of metastases.

Treatment of relapse after allogeneic bone marrow transplantation with unmanipulated G-CSF-mobilized peripheral blood stem cell preparation.

Donor lymphocyte infusions (DLI) are an effective treatment of leukemia relapse after allogeneic bone marrow transplantation. Undesired side-effects are the development of graft-versus-host disease (GVHD) and the occurrence of pancytopenia in some patients. In a pilot study, we investigated if unmanipulated G-CSF-mobilized peripheral blood stem cells which naturally contain large numbers of T lymphocytes (D-PBSC/LI) would be equally effective or even superior than DLI in generating a graft-versus-leukemia reaction (GVL) but could mitigate or prevent the development of pancytopenia. We treated 12 patients with CML chronic phase (n = 5), CML blast crisis (n = 2), AML (n = 2), ALL (n = 1), CLL (n = 1) and multiple myeloma (n = 1). In five patients with acute leukemia or CML blast crisis D-PBSC/LI followed intensive chemotherapy (group A), in seven patients D-PBSC/LI were given without any prior chemotherapy (group B). In group A two patients were evaluable for hematologic toxicity. Leukopenia <1000/microl lasted for 10 and 19 days, and thrombocytopenia <20,000/microl for 11 and 13 days, respectively. In group B leukopenia <1000/microl and thrombocytopenia <20,000/microl was observed in only one patient. Moderate cytopenia developed in four of five evaluable patients. A complete remission could be achieved in all seven patients with CML who all developed acute and/or chronic GVHD. None of the remaining five patients achieved a complete remission despite acute and/or chronic GVHD in two of them. Four patients died from disease progression, one patient from a secondary lymphoma, and one patient as a result of uncontrolled GVHD. In conclusion, D-PBSC/LI is effective in inducing GVL reaction but it does not prevent pancytopenia in each case. It remains unclear if it mitigates the incidence and severity of pancytopenia.

Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.

Tumor contamination of bone marrow (BM) and peripheral blood (PB) may affect the outcome of patients receiving high dose chemotherapy with autologous transplantation of hematopoietic stem cell products. In this report, we demonstrate that replication defective adenoviral vectors containing the cytomegalovirus (CMV) or DF3/MUC1 carcinoma-selective promoter can be used to selectively transduce contaminating carcinoma cells. Adenoviral-mediated reporter gene expression in breast cancer cells was five orders of magnitude higher than that found in BM, PB, and CD34+ cells. Our results demonstrate that CD34+ cells have low to undetectable levels of integrins responsible for adenoviral internalization. We show that adenoviral-mediated transduction of a reporter gene can detect one breast cancer cell in 5 x 10(5) BM or PB cells with a vector containing the DF3/MUC1 promoter. We also show that transduction of the HSV-tk gene for selective killing by ganciclovir can be exploited for purging cancer cells from hematopoietic stem cell populations. The selective expression of TK followed by ganciclovir treatment resulted in the elimination of 6-logs of contaminating cancer cells. By contrast, there was little effect on CFU-GM and BFU-E formulation or on long term culture initiating cells. These results indicate that adenoviral vectors with a tumor-selective promoter provide a highly efficient and effective approach for the detection and purging of carcinoma cells in hematopoietic stem cell preparations.

Hematopoietic Transplant Potential of Unrelated Cord Blood: Critical Issues

To date, hematopoietic stem and progenitor cells from human umbilical cord blood (CB) have been employed in approximately 90 allogeneic (56 sibling and 34 unrelated) matched and mismatched transplantations worldwide with easy and successful restoration of hematopoiesis. Requests for stem cell preparations from CB will continue to increase. Thus, as a pilot study, the examination and standardization of unrelated cord blood-derived stem cell preparations and banking as well as their biologic characterization were initiated. Up to October 1995, a total of 574 samples [mean volume 79 +/- 26 ml, total nucleated cells (NC) 8.5 +/- 5 x 10(8), BFU-E 9.5 +/- 8.6 x 10(5), CFU-GM 5.7 +/- 6.3 x 10(5), CFU-GEMM 1.6 +/- 1.9 x 10(5)] from cord-derived or placental-derived residual blood have been defined by hematologic, immunologic, and microbiologic criteria. These CB samples were collected from the umbilical cord vein immediately after vaginal full-term delivery (n = 450) or cesarean section (n = 124) and stored frozen in liquid nitrogen. Seven percent of all samples collected could not be considered for potential transplants because of volumes < 40 ml. Only 5.0 ml of a CB sample is required for routine laboratory testing, consisting of HLA class I typing, HLA class II typing by sequence-specific oligonucleotide probes (PCR-SSOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony-forming and LTC-IC assays, and CD34+ status. To assess the potential problem of contaminating maternal cells, a PCR was performed on 7 representative samples. During the initial 6 months of the unrelated CB collection program, a median bacterial contamination rate of 18% (20% skin flora species, 80% perineal flora species) was encountered, which has since been reduced to < 1% through practical experience. With regard to viral infections, maternal sera was tested for HBsAg (0.6% positive), anti-HCV (0%), anti-HAV (IgG 18%, IgM 0%), anti-HIV-1-2 (0%), anti-EBV (IgG 98%, IgM 0%), anti-HTLVI-II (0%), anti-CMV (IgG 43%, IgM 0.4%), toxoplasmosis (46%) and syphilis (0%). In addition, all cord blood samples were tested by PCR for CMV infection. With regard to its clinical relevance, it is important that only 0.3% of all the samples were positive for CMV by this sensitive method. This may represent a critical advantage of CB grafts over bone marrow (BM) since, in contrast, > 40% of the unrelated BM donors have been identified to be positive for CMV. An additional advantage of CB is that since 20% of CB samples were collected from ethnic minorities, it appears possible to balance common HLA types and uncommon HLA types represented in this group. In summary, with the extensive practical experience of the obstetric collection team as well as the stem cell-processing laboratory, it appears feasible to obtain a 90% yield of unrelated CB-derived stem cell preparations for banking, which clearly should meet the medical and regulatory qualification criteria required for clinical transplantation. To test the feasibility of hematopoietic transplant potential of unrelated CB for adult patients, ex vivo expansion of CD34+-enriched stem/progenitor cell populations isolated from fresh or frozen CB was attempted in the presence of rh-IL-3, rh-IL-6, rh-EPO, rh-GM-CSF, and rh-SCF with or without fit 3. At varying time points (days 0, 2, 4, 7, 14, 21), the contents of these cultures were analyzed for the numbers of cells, CFC (BFU-E, CFU-GM, CFU-GEMM), and LTC-IC. In this setting, the increase of cells was 200-fold, that of CFC 70-fold, and most importantly that of LTC-IC was 4.5-fold after 7 days in culture in the presence of flt3. In conclusion, LTC-IC derived from CB can be maintained and considerably expanded ex vivo from highly enriched CD34 + CB cell populations from fresh or frozen CB samples.

Purging of peripheral blood stem cell grafts.

The shortage of HLA-matched sibling donors for bone marrow transplant patients has stimulated interest in the use of alternative donors. As a result, there has been a dramatic increase in the use of autologous marrow transplantation, which avoids the complications of graft-versus-host disease, but may deprive the patient of a potentially beneficial graft-versus-disease response and runs the risk of returning occult tumor cells with the graft. There is increasing evidence that these cells may be associated with disease relapse post-transplant, and many methods have been developed for their removal ex vivo. Combinations of negative and positive selection may achieve elimination of tumor cells to the limits of detection of the most sensitive assays currently available. The marked trend toward the use of autologous grafts derived from blood rather than marrow has raised the question as to whether peripheral blood stem cell (PBSC) preparations should be purged of tumor. Data indicate that these grafts generally contain a lower tumor burden, although the stem cell mobilization procedure may recruit tumor cells into the peripheral circulation. Enrichment of CD34+ cells from apheresis products appears, at present, to be less efficient than from marrow and provides at best about a 2-3 log depletion of tumor. This has prompted proposals to follow positive selection by a small-scale purging procedure. Technical issues, such as preprocessing and pooling of collections prior to purging, remain to be addressed. Ultimately, the development of successful purging procedures for PBSC grafts will simply reemphasize the necessity of improving the efficacy of high-dose therapy.

Selective elimination of malignant stem cells using photosensitizers followed by light treatment.

The pros and cons of purging of either bone marrow or peripheral blood stem cell preparations for autologous transplantation for cancer has been debated strongly over the past decade. Recent data implicating the role of minimal residual disease in autografted marrow in cancer relapse have renewed interest in this question. There is a considerable body of literature supporting the possibility that photosensitizer molecules in combination with light might provide a therapeutic window permitting selective elimination of malignant stem cells while sparing those of normal lineage. Molecules of this class are known to be taken up more actively by most malignant cells, and intracellular concentrations are critical in their cytotoxic effect when they are activated by light at an appropriate wavelength. The present paper reviews the observations made over the past decade on a variety of photosensitizers and their effects on hemopoietic progenitors.

The use of the APAAP technique as a rapid indicator of peripheral blood progenitor cell levels

Rapid and sustained engraftment following autotransplantation with peripheral blood stem cells (PBSC) depends on adequate numbers of stem cells and progenitor cells. In this study we have compared the number of myeloid progenitor cells quantitated using the colony forming units-granulocyte macrophage (CFU-GM) clonogenic assay with the number of CD34+ cells estimated both by flow cytometry and by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. We have analysed 15 peripheral blood mononuclear cells (PBMNC) samples from 13 normal subjects and 179 PBMNC from 32 patients undergoing PBSC harvests during the recovery phase of high dose cyclophosphamide chemotheraphy. The number of CD34+ cells measured by the APAAP technique correlated well with the number of CD34+ cells measured by flow cytometry (r = 0.727, p = 0.0001), and also with the number of CFU-GM measured in the clonogenic assay (r = 0.721, p = 0.0001). The APAAP method provides a rapid, reliable measure of progenitor cell levels that can be used to monitor the optimal time to harvest peripheral blood stem cells (PBSC), and to estimate the marrow repopulating ability (MRA) of stem cell preparations used for transplantation.

Design of large-scale separation systems for positive and negative immunomagnetic selection of cells using superparamagnetic microspheres.

The ex vivo selective separation of cells from bone marrow and peripheral blood stem cell preparations is increasingly used as an adjunct to hematopoietic rescue following high-dose therapy for refractory cancer. Immunomagnetic separation, in which the target cells are identified using monoclonal antibodies and separated by attachment to paramagnetic particles and passage through a magnetic field, is widely used for both negative and positive cell selection. In this paper, we discuss the factors that should be considered when developing a magnetic separation device for purging tumor cells and selecting stem cells from bone marrow using superparamagnetic microspheres.

Autologous graft engineering.

Autologous transplantation of bone marrow or peripheral blood stem cell preparations is now a widely used form of rescue from the myeloablative effects of high-dose therapy for malignant disease. The success of this procedure is affected by numerous factors associated both with management of the patient and the quality of the graft. In this review, approaches are described to maximize the success and utility of this therapeutic approach.

Transplantation of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease.

Hematopoietic stem cell defects are thought to be involved in the etiopathogenesis of systemic autoimmune disease. Positively selected, stem cell-enriched populations of wheat germ agglutinin-positive (WGA+) low-density bone marrow and fetal liver cells from normal and autoimmune-prone mice were used to determine whether reciprocal transplantation of stem cells between normal and autoimmune-prone mice inhibits or causes development of autoimmune disease. NZB recipients of DBA/2 stem cell populations analyzed greater than 100 days after bone marrow or fetal liver cell transplantation showed decreased levels of anti-DNA antibodies and decreased glomerular lesions when compared with nontreated NZB mice or NZB recipients of NZB stem cell preparations. Female DBA/2 recipients of WGA+ NZB bone marrow cell or fetal liver cell transplants exhibited elevated serum autoantibody levels and developed glomerular lesions characteristic of NZB mice when analyzed greater than 100 days after transplantation. These pathological disturbances were not observed in DBA/2 recipients of DBA/2 stem cell preparations. The data indicate that WGA+ stem cells from autoimmune-prone NZB mice contain the genetic defects responsible for the development of systemic autoimmune disease.

A new technique for peripheral stem cell separation

Using a Haemonetics V50.1 cell separator we compared the new lymphocyte-surge-program for peripheral stem cell preparation with the conventional combined buffy coat and Ficoll techniques. Eleven surge and four conventional separations were performed in 15 healthy cytapheresis donors who gave informed consent to the procedure. Using the conventional methods, mean recovery of peripheral blood mononuclear cells (PBM) was 55% in a preparation time of 80 minutes for the buffy coat and 25 minutes for the Ficoll step. Eighty percent of the PBM loss occurred during the buffy coat step, and 20% during the Ficoll procedure. In a preparation time of 105 minutes, 2500 mL peripheral blood could be processed without flow or citrate problems. The donors' red cell losses were about 100 mL packed red cells. Granulocyte depletion was about 98%, red cell depletion higher than 99% (hematocrit 7 PBM) in both separation techniques. Therefore, both techniques seem to be adequate for T-cell depletion and preparation of bone marrow for cryopreservation. Since preparing bone marrow for purging techniques requires extreme stem cell purity, the surge technique may be less suitable for those purposes due to the awkward problem of considerably higher red cell contamination, despite a shorter preparation time.

Collection, storage, and use of hematopoietic stem cells from peripheral blood

Complete hematopoietic reconstitution using nonleukemic peripheral blood mononuclear cells has yet to be achieved in humans. Significant advances have been made in in vitro quantification of putative stem cells, in animal models of the reconstitutive process, and in the collection, processing, and storage techniques for stem cell preparations. These may eventually lead to use of hematopoietic stem cells from peripheral blood for both allogeneic and autologous bone marrow reconstitution in a variety of clinical situations.

Development of cytotoxic T lymphocyte precursors in the absence of the thymus.

AbstractReconstitution of adult thymectomized and lethally irradiated C3H or (C 3 H × DBA/2) F1 mice with syngeneic adult bone marrow cells or 18 to 20‐day‐old fetal liver cells resulted almost regularly in the generation of large numbers of cytotoxic T lymphocyte precursors (CTLP) against trinitrophenylated C3H cells and three types of allogeneic stimulator and target cells. The responses against 2,4,6‐ trinitrophenylated cells were restricted and in so far typical for cytotoxic T lymphocytes.The failure of bone marrow cells from athymic C 3 H mice to reconstitute significant cytotoxic activity indicated that the CTLP had developed from “cytotoxic T lymphocyte preprogenitors” (CTLPP) which were derived from the donor and which had already been under thymic influence. The stem cell preparations in themselves contained frequencies of mature CTLP far too low to account for the reactivity and CTLP frequencies in the reconstituted mice. This fact implied that the CTLPP proliferated extensively and/or matured in the recipient in the absence of the thymus.Individual mice failed to respond to one or more of these stimulator cells. The failure of individual mice to respond was randomly expressed for the four specificities even when the mice were reconstituted with the same stem cell preparation suggesting that CTLPP with defined specificity and low frequency (about 1/108 in fetal liver) were contained in the stem cell preparation and transferred randomly into the irradiated recipients. This would imply that one CTLPP gives rise to 5 × 103–20 × 103 CTLP within 5 weeks.The data suggest that the peripheral pools, at least of certain T cell subclasses, may be maintained largely by post‐thymic proliferation of CTLPP.

"Early T cells" and "late T cells"; suggestive evidence for two T cell lineages with separate developmental pathways.

Peripheral lymphocytes from mice have been analyzed by a combination of cell electrophoresis and size distribution analysis and the data have been processed with a computer into two-dimensional distribution patterns (fingerprints). The fingerprints of lymph node cells revealed the existence of at least three major classes of small lymphocytes. Cells with similar physical properties were found in the spleen and thoracic duct lymph. The data also allowed calculation of the quantitative ratios of the three cell types. In the normal mouse, the two electrophoretically faster cell classes represent essentially two subclasses of T cells. The quantitative ratios are here always in agreement with the known percentages of B and T cells. Lethally irradiated mice that have been reconstituted with syngeneic bone marrow or fetal liver cells often lack one of the T cell subclasses. This has two important implications. 1) It indicates that the widely used syngeneic or allogeneic bone marrow chimeras may be incomplete in respect to their T cell repertoire and that experiments with these models should be interpreted with caution. 2) The deficiency was found to correlate strictly with a deficiency in a physically defined subset of small cortical thymocytes that has been described previously. This correlation suggests strongly that the fast peripheral T cells and the "early small cortical thymocytes" represent two developmental stages of a distinct cell lineage ("early T cell lineage"), while the thymic pool of "late small cortical thymocytes" gives rise to electrophoretically slow peripheral T cells, which may be called "late T cells). The possibility that both T cell lineages are derived from different classes of prethymic stem cells in the reconstitlting stem cell preparation is discussed. The proportion of "early T cells" in the lymph nodes decreases with age. The ratio of "late" and "early T cells" is similar in the spleen and in the lymph nodes, and both cell classes contain a significant proportion of cells that are not affected by the early effects of adult thymectomy. Thus, they do not correspond to T1 and T2 cells.