Michael J. Alcorn

CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens

We have previously demonstrated that CD34+ cells, selected from peripheral blood progenitor cells (PBPC), can be expanded in ex vivo culture and can be infused in tandem with unmanipulated PBPC with little or no toxicity. In this study, four patients (two non-Hodgkin’s lymphoma (NHL), two multiple myeloma (MM)) received myeloablative conditioning prior to stem cell rescue using ex vivo expanded cells alone. The two patients with NHL received cyclophosphamide and total body irradiation (CY/TBI) and the two patients with MM, busulphan and melphalan (Bu/M). One case received an inadequate CFU-GM dose, despite expansion, and in one case the expanded cells were contaminated. No definitive conclusions may therefore be drawn concerning engraftment in these two cases. However, the other two cases received high doses of committed progenitors. Following infusion of the expanded material, all four patients failed to show sustained neutrophil engraftment and none showed evidence of platelet engraftment. Back-up, unmanipulated PBPC were therefore infused on days 14, 34, 32 and 28 and subsequently all four cases achieved satisfactory engraftment of both neutrophils and platelets. In conclusion, we feel that, CD34+ cells, expanded ex vivo using the conditions described in this report, may not provide durable engraftment following fully myeloablative conditioning.

The Number of CD34+ Cells Mobilized into the Peripheral Blood Can Predict the Quality of Subsequent Collections

PBPC were mobilized using a variety of chemotherapy regimens plus G-CSF in a group of 126 consecutive patients. Data are presented that show a close correlation between the number of CD34+ cells mobilized into the peripheral blood (PB) and the number of CD34+ cells subsequently collected by leukapheresis (R = 0.904). On the basis of this correlation, a regression formula was calculated that could give an estimate of the total number of CD34+ cells likely to be collected by leukapheresis from a given number of CD34+ cells per microliter PB. An easy-to-read table has been compiled to show how this type of analysis can be applied to predict the likely dose of CD34+ cells that will be obtained by leukapheresis over a wide range of patient weights.

Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).

BACKGROUND It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery). METHODS In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection). RESULTS The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%). DISCUSSION Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.

CD34 Selection and EX Vivo Expansion of Haemopoietic Progenitor Cells: A Review of Laboratory Methodology

The CD34 antigen is expressed on haemopoietic stem and progenitor cells. A number of strategies have been developed which allow the selection and purification of CD34(+) cells from bone marrow, peripheral blood, and umbilical cord blood. Transplantation studies have amply demonstrated that rapid and durable engraftment can be achieved following reinfusion of selected CD34(+) cells. More recently, techniques have become available which can produce extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most popular method involves a simple liquid suspension culture system supplemented with a range of cytokines. The degree of expansion and, indeed, the types of cells produced can be significantly influenced by culture conditions like the choice of cytokines, duration of culture, starting cell concentration, and type of culture vessel. Despite many laboratory investigations, there have been few clinical trials using ex vivo expanded cells. Although it has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity, there is no evidence to date that these culture systems sustain sufficient numbers of haemopoietic long-term repopulating cells to secure durable engraftment following myeloablative therapy. Clearly, the major goal is to define culture conditions which will produce true stem cell expansion.

CML leukapheresis products can be enriched for CD34 + cells and simultaneously depleted of CD15 + cells using a simple Ab cocktail

BACKGROUND CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells. METHODS Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured. RESULTS The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained. DISCUSSION A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.

INTERLEUKIN-10 Production and monocyte maturity in g-csf-mobilized peripheral blood stem cell transplants correlate with graft-versus host disease

Abstract Despite an increased number of donor T cells in peripheral blood stem cell (PBSC) collections mobilized with G-CSF, the incidence of severe acute graft-versus-host disease (GVHD) is not increased compared to bone marrow transplant (BMT) recipients. However, the incidence of chronic GVHD has been reported to be higher with PBSC than BMT, and this may be due to the balance of cytokines produced by the graft. Here we have examined monocyte phenotype and cytokine production in the PBSC grafts of 27 patients that have undergone allogeneic PBSC grafting for haematologic malignancy. Monocytes in PBSC grafts appeared to be immature with a significantly lower CD14 expression by CD64 + cells within 22/27 PBSC collections. The presence of immature monocytes was significantly correlated (p = 0.0361) with absent or low acute GVHD (grade 1), while monocytes of a more normal phenotype were contained in the grafts of those developing severe acute GVHD (grades 2–4). Furthermore, monocytes in PBSC grafts showed a significantly increased spontaneous production of IL-10 compared to non-mobilized controls (p

Detection of IL-3 and g-csf mRNA and protein in CD34+ progenitor cells and serum from cml patients

Abstract Our previous studies demonstrated that primitive subsets of leukaemic cells isolated from patients with chronic phase CML exhibit growth factor independent proliferation, in both single cell and bulk cultures, which is at least partially dependent on autocrine production of bioactive IL-3 and to a lesser degree G-CSF (PNAS 96; 12804, 1999). In those studies ∼ 85% of the cases investigated were CML patients in whom the stem cell compartment (LTC-IC) was already predominantly Ph + . Since such cases represent less than 10% of newly diagnosed patients, it was of interest to examine IL-3 and G-CSF mRNA expression in CD34 + progenitor cells and any corresponding protein production in serum in a larger series of CML patients independent of the Ph status of their LTC-IC compartment. CD34-enriched lineage-negative cells were isolated using STEMSep TM from leucapheresis samples obtained at diagnosis from chronic phase patients and viable, propidium iodide (PI)-negative, annexin V-negative, CD34 + cells then isolated by FACS. Total RNA was extracted from ≥ 5 × 10 4 such cells and c-ABL, BCR-ABL, IL-3 and G-CSF expression examined using a 2-step RT-PCR procedure. 8 of the 10 CML samples tested to date are positive for IL-3 expression. Using sensitive ELISA kits, G-CSF was detected at significantly lower levels in the sera of CML patients (7.2 ± 2.9 pg/ml, n=13) than in normal controls (24.5 ± 3.2 pg/ml, n=9, p=0.001), with levels correlating inversely to the WBC. In untreated patients with elevated WBC (45–166 × 10 9 /L) G-CSF levels proved to be undetectable. In patients on hydroxyurea, in whom the WBC was controlled ( 9 /L), levels were low but above the threshold for detection (2–11 pg/ml). Of interest, in patients who had achieved a complete cytogenetic remission following either allogeneic BMT or interferon therapy, levels were within the normal range. These findings support the proposed negative feedback mechanism between peripheral neutrophil count and serum G-CSF levels. IL-3 was detected in sera from 1 of 9 normal controls and 5 of 29 CMLs (range 1–35 pg/ml), 3 of whom were in accelerated phase. These results confirm that IL-3 mRNA expression in the progenitor compartment is typical of the majority of cases of CML. However, this does not necessarily lead to detectable levels of this growth factor in the circulation suggesting its effects in vivo may be largely confined to extravascular regions of the marrow and other tissues that are infiltrated by CD34 + leukaemic cells.

Spontaneous reactivation of primitive quiescent cml cells is associated with upregulation of IL-3 expression

Abstract CML is a myeloproliferative disease of stem cell origin. Cell cycle active anti-neoplastic agents fail to eradicate the leukaemic clone suggesting the presence of a rare subset of quiescent leukaemic stem cells. By examining the gene expression of such cells and on their entry into cell cycle, the mechanisms supporting their viability and quiescence may be determined. Candidate lin − CD34 + haemopoietic stem cells were separated into cycling (G 1 SG 2 M) and non-cycling (G 0 ) populations by FACS and cultured separately, in serum free medium with or without added growth factors (GF). Gene expression analysis by RT-PCR on FACS-sorted viable (Propidium iodide − /Annexin-V − ) cells was carried out at baseline and on days 4, 8 and 12. In parallel, individual G 0 cells were automatically sorted into 96-well plates and cultured with or without GF. At baseline, quiescent BCR-ABL + cells expressed high levels of CD34, p21 and cyclin D2, low levels of Ki-67, cdc25 and TGF-β and very few contained either IL-3 or G-CSF transcripts. In comparison, cycling BCR-ABL + cells expressed similar levels of CD34 and cyclin D2, less p21, higher levels of Ki-67, cdc25 and TGF-β, and the majority were positive for IL-3 and G-CSF. Following culture in the absence of added GF, (and in association with entry into cell cycle, clone formation from single cells, upregulation of Ki-67 and cdc25), IL-3 and G-CSF transcripts could be detected, in association with continued BCR-ABL and CD34 expression, in the progeny of quiescent CML cells. Thus, in CML patients, quiescence is accompanied by a lack of constitutive IL-3 or G-CSF expression (in spite of the continued presence of BCR-ABL and CD34 mRNA), and is spontaneously reversible (in the absence of GF) in association with an upregulation of IL-3 and/or G-CSF expression. Under conditions of low cytokine concentrations these cells would be predicted to enjoy a proliferative advantage over their quiescent Ph − /BCR-ABL − counterparts and may therefore be of potential physiological relevance in CML.