• CD34 cell separation: from basic research to clinical applications.

      Nadali, Gianpaolo; De Wynter, Erika A; Testa, Nydia G; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (1995)
      Advances in the immunological identification of primitive haematopoietic cells have led to the development of various techniques for their characterisation and purification. The expression of the CD34 antigen by the stem cell compartment has been exploited for these purposes. Separation systems capable of isolating CD34+ cells on a large scale are finding use in the clinic. Areas of interest overlap for both researchers and clinicians, and efforts to mobilise, characterise, purify and transplant these cells continue. Different CD34 purification systems are reviewed, focusing on their possible applications in different research fields. Clinical results of CD34+ selection and the possible future applications of this technology in the clinic are also reported.
    • Effect of addition of FLT-3 ligand and megakaryocyte growth and development factor on hemopoietic cells in serum-free conditions.

      Rossi, Barbara; Zanolin, Elisabetta; Vincenzi, Carlo; Diani, Franco; Pizzolo, Giovanni; De Wynter, Erika A; Nadali, Gianpaolo; Department of Clinical and Experimental Medicine, Section of Hematology, University of Verona, Italy. (2004-08)
      The aim of this study was to clarify the mechanisms that regulate hematopoietic cell expansion in vitro by identifying defined culture conditions. We report the results of experiments with CD34(+) cells from cord blood (CB, n = 13), bone marrow (BM, n = 4), and mobilized peripheral blood stem cells (PBSC, n = 5) using two combinations of cytokines: (A) granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), stem cell factor (SCF), erythropoietin (EPO), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-b) and (B) combination A plus FLT3 ligand (FL) and megakaryocyte growth and development factor (PEG rhMGDF). Cultures of immunoselected CD34(+) cells were performed in serum-free liquid medium without serum substitutes. The area under the curve (AUC) obtained by plotting the logarithm of the total number of viable cells, CD34(+) cells, and CFC per well, toward the week of culture was used as an index of cell expansion. With CB, a significant difference was obtained between the two combinations of cytokines with regard to the total number of viable cells, GM-CFC, and CD34(+) cells. The difference between the two combinations of cytokines obtained with BM was significant with respect to the total number of viable cells and CD34(+) cells but not for the erythroid and myeloid progenitors. When CD34(+) cells from peripheral blood stem cells (PBSC) were cultured in presence of the two combinations of cytokines, the difference in terms of AUC was not statistically significant. Our data indicate additional effects in terms of proliferation and expansion of hematopoietic cells in serum-free conditions when FL and polyethylene glycol (PEG) rhMGDF are included in culture and suggest a differential activity of these cytokines on cells from different hematopoietic sources.
    • Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

      De Wynter, Erika A; Nadali, Gianpaolo; Coutinho, Lucia H; Testa, Nydia G; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom. (1996-09)
      CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.
    • Multicentre European study comparing selection techniques for the isolation of CD34+ cells.

      De Wynter, Erika A; Ryder, W David J; Lanza, F; Nadali, Gianpaolo; Johnsen, H; Denning-Kendall, P; Thing-Mortensen, B; Silvestri, F; Testa, Nydia G; Paterson Institute for Cancer Research, Withington, Manchester, UK. (1999-06)
      Primitive haemopoietic cells are required for studies in both the clinical and research fields and a number of systems have been developed to facilitate isolation of these haemopoietic cell populations. We have analysed the results from several European centres using positive selection of CD34+ cells from haemopoietic tissues (n = 110). Four selection techniques including immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) and submicroscopic magnetic beads (MACS) were used and the selected CD34+ cells were assessed for purity, yield and enrichment of colony-forming cells (CFC). The mean purities for all samples ranged from 68.4-78.4% for MACS, 33.9-69.9% for Dynabeads, 46.9-66.8% for Ceprate LC and 43.2-65% for Baxter Isolex 50. Yields were variable with all techniques. On average CFC enrichment using the immunoaffinity columns was greater than that observed for the other systems. Some techniques appear to be problematic and may require further expertise to improve the results. Nevertheless, the study demonstrates that highly purified CD34+ cells can be isolated from various haemopoietic sources, though yield and CFC enrichment varies significantly depending on the technique selected. This extends our previous report indicating that not all selection methods generate similar results and that there are differences in the purity, number and colony-forming ability of the cells recovered.
    • Regulation of the proliferative potential of cord blood long-term culture-initiating cells (LTC-IC) by different stromal cell lines: implications for LTC-IC measurement.

      Nadali, Gianpaolo; De Wynter, Erika A; Perandin, F; Tavecchia, L; Vincenzi, C; Ambrosetti, A; Fornalè, M; Perona, Giuseppe; Pizzolo, Giovanni; Testa, Nydia G; et al. (1998-12)
      BACKGROUND AND OBJECTIVE: Long-term culture-initiating cells (LTC-IC) are the best available approximation to an in vitro assay of stem cells in humans although they still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord blood (CB) is rich in hemopoietic progenitor cells, as measured by clonogenic assays and contains stem cells capable of reconstituting the marrow after ablation in clinical transplantation. We evaluated the influence of culture conditions on the in vitro behavior of LTC-IC from CB. DESIGN AND METHODS: LTC-IC were evaluated in long-term cultures, comparing two types of murine stromal cell lines: M2-10B4 and M2-10B4 transfected with cDNAs for human G-CSF and IL-3. RESULTS: Two and five fold higher numbers of terminally differentiated cells were produced during nine weeks of culture of CB mononuclear or CD34+ cells respectively, in cultures containing a M2-10B4 IL-3 G-CSF cell line compared to cultures containing the parental cell line. Likewise, a higher number of colony-forming cells (CFC) were detected in the supernatant of cultures with the transfected cell line. In contrast, the number of CFC generated within the stromal layer, after 5 or 9 weeks of culture, was significantly higher in cultures on M2-10B4 cells than those on M2-10B4 IL-3 G-CSF. INTERPRETATION AND CONCLUSIONS: Our results show that the proliferative capacity of CB LTC-IC can be strongly influenced by culture conditions and that the frequency of LTC-IC estimated using these cell lines as stromal support is not identical.