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.

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+ cells can be selected efficiently from cryopreserved peripheral blood progenitor cells and can retain their proliferative potential.

Hematopoietic stem and progenitor cells express the CD34 antigen. Techniques have been developed that enable purified populations of CD34+ cells to be selected from hematopoietic tissues. These selected CD34+ cells have several potential applications, including CD34 selection to obtain a tumor purging effect in autologous transplantation studies and using CD34+ cells as the starting cells for ex vivo expansion studies and as a vehicle for gene transduction protocols. We have investigated the feasibility of using cryopreserved peripheral blood progenitor cells (PBPC) for CD34 selection. Cells could be recovered from cryopreservation with good yields and high viability. After CD34 selection, the final product was, on average, 84% pure, with a recovery of 54%. These cells retained extensive proliferative potential, as demonstrated by ex vivo expansion culture. We believe that cryopreserved PBPC could be thawed, and CD34+ cells could be selected and used for transplantation following high-dose chemotherapy.

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.

Efficient isolation of human CD34 positive hemopoietic progenitor cells by immune panninga.

In this study we have assessed the use of soybean agglutinin (SBA) and CD34 microcellector devices for the selection of CD34 positive hemopoietic progenitor cells. Burst forming unit-erythroid (BFU-E), colony forming unit-granulocyte/macrophage (CFU-GM) and the recently developed multipotential human colony forming unit-type A (CFU-A) clonogenic assays were used to measure progenitor numbers in the starting mononuclear cell (MNC), the SBA negative, the nonadherent CD34 negative and the adherent CD34 positive fractions during panning. CFU-A progenitors were present at a relatively high incidence in the MNC fraction (220 per 10(5) MNC) and were enriched 15-fold in the adherent CD34 positive fraction. This progenitor incidence and enrichment were similar to those of CFU-GM and BFU-E. The mean recovery for CD34 positive cells was 2.3 x 10(6) cells per marrow aspirate. Analyses by flow cytometry demonstrated that 1-5% of input MNC were CD34 positive, that the purity of the CD34 fraction was approximately 80% and that the calculated recovery for CD34 positive cells was 61%. Recoveries for CFU-GM, BFU-E and CFU-A were between 18 and 40%. CFU-A progenitors were found exclusively in the adherent CD34 positive fraction, whereas a significant proportion of both CFU-GM and BFU-E were present in the nonadherent CD34 negative fraction. We propose that the Applied Immune Sciences (AIS) flasks preferentially bind the cells which express CD34 most strongly and that this is reflected in the finding of primitive CFU-A only in the CD34 positive fraction, with lineage-restricted progenitors found in both CD34 positive and negative fractions. This hypothesis is strengthened by data on long-term bone marrow cultures in which the CD34 positive fraction is better able to maintain output of CFU-GM compared with the CD34 negative fraction. In conclusion, relatively pure populations of CD34 positive cells may be rapidly and efficiently isolated from bone marrow samples with good recovery. The isolated cells show enhanced colony forming capacity in standard clonogenic assays and in the multipotential CFU-A assay.

Contrasting effects of rh-MIP-1 alpha and TGF-beta 1 on chronic myeloid leukemia progenitors in vitro.

In chronic myeloid leukemia (CML) an abnormality at the stem cell level results in unregulated expansion of myeloid progenitors. The mechanism underlying this uncontrolled proliferation remains unclear. An in vitro clonogenic assay which detects the human counterpart of the murine colony forming unit (CFU) CFU-A/CFU-S day 12 was described in a report of our recent findings. CML bone marrow samples were found to proliferate in the CFU-A assay, producing colonies morphologically indistinguishable from normal controls. The bcr/abl transcripts were sought in the RNA from individual colonies using the polymerase chain reaction (PCR). For the five CML samples tested to date, the majority of CFU-A colonies at diagnosis or in early chronic phase were found to be bcr/abl positive. For normal controls both macrophage inflammatory protein-1 alpha (MIP-1 alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibited the proliferation of CFU-A colonies when directly added to the assay. In contrast, CML progenitors responded normally to TGF-beta 1, but showed no response to MIP-1 alpha. In suicide assays, for five normal bone marrow samples, CFU-A progenitors induced into S-phase returned to a quiescent state after treatment with MIP-1 alpha. CML progenitors demonstrated inherently high cycle status which showed no definite response to MIP-1 alpha. However, TGF-beta 1 resulted in quiescence of CML progenitor cycling. In conclusion, the primitive progenitors from CML samples were inhibited normally by TGF-beta 1 but showed no response to MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Men, women, and religion: stress within leadership roles.

Stress in religious leaders was investigated by administering to 250 priests, ministers, seminarians, nuns, and brothers the specially devised Religion and Stress Questionnaire and the Osipow and Spokane Occupational Environment Scales, Personal Strain Questionnaire, and Personal Resources Questionnaire. In-depth interviews were also conducted with 10% of the subjects. Religious leaders experienced lower overall occupational stress and personal strain and evidenced more personal resources than did the normative population. ANOVAs showed ministers to have the highest overall occupational environment stress and vocational strain and next to the lowest scores in overall personal resources of the five groups of religious professionals. In terms of sensitivity to the stress that women in seminary and ministry experience, male and female seminarians and ministers were most aware of these issues, with priests (males only), nuns, and then brothers (in descending order) sensitive to these concerns.