Biomedical subjects

D C Dooley

Publications and source records attributed to D C Dooley.

At least 19 recordsLinked to original sources

PubMed2001

Transforming growth factor-beta(1) induces apoptosis in CD34(+)CD38(-/low) cells that express Bcl-2 at a low level.

OBJECTIVE: Transforming growth factor-beta(1) (TGF-beta(1)) strongly inhibits the proliferation and differentiation of primitive CD34(+)CD38(-) hematopoietic cells. In contrast, Flt3 ligand (FL) is a positive effector of CD34(+)CD38(-/low) cell proliferation. Because apoptosis plays a critical role in hematopoietic development, TGF-beta(1) and FL were analyzed as possible modulators of apoptosis. Specifically, this report examined expression of apoptotic promoters Bax and Bad and apoptotic inhibitors Bcl-2 and Bcl-x (all members of the Bcl-2 protein family). Protein levels were determined in fresh and cultured CD34(+)CD38(+) cells and CD34(+)CD38(-/low) cells with and without treatment with TGF-beta(1) and FL. MATERIALS AND METHODS: Cells fractions were purified by sorting CD34(+)-enriched mononuclear cells from mobilized peripheral blood. Expression of Bcl-2, Bcl-x, Bax, and Bad and the extent of apoptosis were determined by flow cytometric analysis of freshly isolated cells and cells cultured with TGF-beta(1) and FL effectors. RESULTS: TGF-beta(1) reduced CD34(+)CD38(+) cell expansion and arrested cell division. Inhibition of growth was not accompanied by an increase in apoptosis. In CD34(+)CD38(-)(/low) cells, serum TGF-beta(1) and added TGF-beta(1) inhibited cell growth and significantly increased apoptotic cell death. Freshly isolated CD34(+)CD38(+) and CD34(+)CD38(-/low) cells expressed Bcl-2 at similar low levels. However, after 3 days, Bcl-2 expression was markedly higher in cultured CD34(+)CD38(+) cells. TGF-beta(1) significantly increased Bax expression in both fractions after 3 days cultivation (p = 0.0034). Thus, addition of TGF beta-1 further reduced the already low Bcl-2:Bax ratio in CD34(+)CD38(-/low) cells. CONCLUSIONS: Compared to CD34(+)CD38(+) cells, CD34(+)CD38(-/low) cells were slow to up-regulate expression of Bcl-2 during ex vivo culture. TGF-beta(1) up-regulated Bax expression by both CD34(+)CD38(+) and CD34(+)CD38(-)(/low) cells and promoted apoptosis in the latter fraction. This suggests that the preferential induction of apoptosis in primitive cells by TGF-beta(1) may be due to its further reduction of the Bcl-2:Bax ratio.

ADP-ribosyl Cyclase↗

PubMed2000

Cellular and molecular aspects of human CD34+ CD38- precursors: analysis of a primitive hematopoietic population.

Hematopoiesis is a complex, highly regulated process in which a small number of primitive stem cells produce all the mature blood and immune cells required by an animal throughout its life. In this review, we summarize current understanding of human CD34+ CD38- cells, a small subclass of hematopoietic cells, which is enriched for primitive precursors, including stem cells. This review emphasizes functional, molecular and immunophenotypic characteristics of the cells and includes some of the factors which are believed to regulate the survival, growth and differentiation of this important class of cells.

ADP-ribosyl Cyclase↗

PubMed1999

Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.

ADP-ribosyl Cyclase↗

PubMed1997

Flt3 ligand enhances the yield of primitive cells after Ex vivo cultivation of CD34+ CD38dim cells and CD34+ CD38dim CD33dim HLA-DR+ cells.

Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma-free culture system. Towards that end, four CD34+ subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit-granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- +/- 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33(dim) HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim cells and CD34+ HLA-DRdim cells after 5 to 12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34+ CD38dim or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P < . 05). For HLA-DR+ cells, LTCIC recoveries averaged 214% +/- 87% of input with FL and 24% +/- 16% without FL. In contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34+ cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34+ cells, and CFU-GM and enhances the pool of early CD34+ CD33(dim) cells, CD34+ HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool.

ADP-ribosyl Cyclase↗

PubMed1995

Transforming growth factor beta 1 inhibits expression of the gene products for steel factor and its receptor (c-kit).

Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF-beta 1 has been shown to repress human and murine leukemic cell and murine lin- bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF-beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin- hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo.

Acute Disease↗

PubMed1995

Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long-term stromal cultures.

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary long-term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose-dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma-free long-term cultures, neither factor affected CFU-GM expansion. Basic FGF slightly enhanced granulocyte-macrophage colony forming unit (CFU-GM) cloning efficiency in short-term agarose culture. Basic FGF did not reduce the levels of interleukin-6 (IL-6), GM-CSF, or G-CSF released by steady state or IL-1-stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF-beta 1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified.

Bone Marrow↗

PubMed1993

Constitutive expression of steel factor gene by human stromal cells.

Steel factor (SF), the ligand for c-kit, is an essential regulator of normal hematopoiesis, melanogenesis, gametogenesis, and mast-cell growth and development. Hematopoietic stromal cells are important sources of SF, because inactivation of SF in mice results in defects in the support function of hematopoietic stromal cells. To identify specific cells that produce, and factors that govern the expression of the different isoforms of SF in human hematopoiesis, we quantified levels of SF mRNA and membrane-bound protein in human stromal cells before and after exposure to recombinant human interleukin (IL)-1 alpha, a cytokine known to induce the expression of a variety of hematopoietic growth factors. In addition, because stromal cells in longterm bone marrow cultures (LTBMC) are supportive of hematopoietic progenitor cell survival in vitro, while umbilical vein endothelial cells (EC) and diploid fibroblasts (DF) are not, we also sought to test the hypothesis that SF gene expression would differ in cells from LTBMC when compared with EC or DF. Using reverse-transcription polymerase chain reaction amplification (RT-PCR), ribonuclease protection assays (RPA), and Northern blot analysis, SF was found to be constitutively transcribed in EC, DF, and LTBMC. IL-1 alpha neither induced accumulation of SF mRNA nor altered the ratio of exon 6+ to exon 6- transcripts in these stromal cells. By Northern blot analysis, the predominant SF mRNA species was shown to be 5.6 kb; a minor population of 3.6 kb was also found. Low levels of membrane-bound SF protein were found to be constitutively expressed by all three types of stromal cells, and were not regulated by IL-1 alpha. We conclude that the unique capacity of LTBMC to support in vitro hematopoiesis, when compared with EC or DF, cannot be explained on the basis of qualitative or quantitative differences in SF gene expression in these cells.

Alternative Splicing↗

PubMed1993

Autologous transplant programs in community hospitals: role of centralized stem cell processing laboratories.

Transplantation of bone marrow and peripheral blood stem cells was initially limited to large, research-oriented institutions. General acceptance of autologous transplantation has grown rapidly in recent years and there is now interest in the establishment of autologous transplant programs in community hospitals. However, the lack of facilities to process and cryopreserve stem cells can be a major obstacle to small transplant programs. In response to this problem, we suggest that a centralized stem cell processing facility can offer the required services to several hospitals within a metropolitan area. The establishment of processing facilities within currently existing blood banks and blood centers is a logical extension of their current roles in hematotherapy. In this paper, we describe the operation of one such centralized processing facility.

Blood Banks↗

PubMed1992

Characterization and functional expression of genomic DNA encoding the human lymphocyte type n potassium channel.

Voltage-gated potassium channels play important functional roles in the development and maintenance of human lymphocyte functions. One such channel, known as the type n channel, has been well defined in human T cells and exhibits unique functional properties that distinguish it from other species of potassium channels. We report the characterization of a human genomic DNA clone, HGK5, encoding a 523-amino-acid potassium channel protein encoded by an open reading frame on a single exon. RNA transcribed in vitro from HGK5 genomic DNA directs expression of functional voltage-dependent potassium currents in Xenopus oocytes. The functional characteristics of the expressed channels are strikingly similar to those of the type n channel on human T lymphocytes. This, together with the presence of significant levels of HGK5 mRNA in human T lymphocytes, supports the notion that HGK5 encodes the human type n voltage-gated potassium channel. The effects of concanavalin A treatment on HGK5 mRNA levels in cultured human T lymphocytes was also examined. Mitogenic concentrations of concanavalin A induced a time-dependent decrease in HGK5 mRNA levels, suggesting that previously observed increases in potassium current density following concanavalin A treatment of human T lymphocytes are not due to increased transcriptional activity of the type n potassium channel gene.

Amino Acid Sequence↗

PubMed1992

Detection and quantitation of long-term culture-initiating cells in normal human peripheral blood.

The objective of this study was to utilize the Dexter long-term culture system to characterize and quantitate early hematopoietic cells in normal human blood. Peripheral blood mononuclear cells were depleted of T cells. B cells, and monocytes, yielding a population (null cells) highly enriched for hematopoietic precursors. One to 2 x 10(7) null cells containing an average of 5.4 x 10(4) granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) were cultured on irradiated allogeneic marrow stromal layers in T-25 flasks. Nonadherent progenitors were detected for an average of 6.8 +/- 0.7 (standard error of the mean) weeks and exceeded 1 x 10(4) per flask for 4 weeks. Null cells were treated with 5-fluorouracil (5-FU) to determine whether long-term growth was due to survival of CFU-GM or to maturation of more primitive precursors. When 5-FU treatment killed greater than 99% of CFU-GM, an increase in progenitors was observed after 12 days of cultivation with stroma. CFU-GM cloning efficiency of 5-FU-treated cells peaked after 24 days of incubation, whereas controls peaked after 7 days. The total number of nonadherent CFU-CM in 5-FU-treated and control cultures converged during the 5-8 weeks of incubation. The frequency of long-term culture-initiating cells (LTCIC) in peripheral blood was determined by limiting dilution analysis in micro-stromal cultures. In two donors, the frequencies of LTCIC were 6.3 and 19.6 per 1 x 10(5) null cells. The CFU-GM:LTCIC ratios were 12.6:1 and 12.8:1. We conclude that normal peripheral blood contains primitive hematopoietic precursors demonstrable as 5-FU-resistant precursors of CFU-GM or as LTCIC. The frequency of the latter is 13-fold lower than day-14 CFU-GM.

B-Lymphocytes↗

PubMed1991

Methods for measuring a 6 log10 white cell depletion in red cells.

Methodology is presented for enumerating very low concentrations of white cells (WBCs) in red cells (RBCs) by two separate measurement techniques. Both techniques rely on the method of harvesting WBCs from a 300- to 350-mL unit of RBCs and concentrating them to a volume of approximately 0.5 to 1.0 mL, which is equivalent to a WBC concentration of approximately 550 to 1. The WBC separation and concentration steps require less than 3 hours to complete, and multiple RBC units can be processed in parallel. Cell counting is carried out in a fluorescence hemocytometer or by a modified cytospin technique. As few as 1000 WBCs in a unit of RBCs, which corresponds to a more than 6 log10 WBC depletion, can be measured without reaching the sensitivity limit of either technique (800 and 200 WBC/unit, respectively). The harvesting method and counting techniques are relatively simple and inexpensive.

Acridine Orange↗

PubMed1990

Transforming growth factor-beta 1 and T-lymphocytes modulate the growth of human peripheral blood progenitor cells.

This study examined the effects of transforming growth factor-beta 1 (TGF-beta 1) and autologous T-lymphocytes on the growth of day-14 granulocyte-monocyte progenitor cells (CFU-GM). Mononuclear cells from human peripheral blood were depleted of accessory cells, yielding "B/null" or "null" cell populations enriched 17-fold and 94-fold, respectively, for CFU-GM. Addition of TGF-beta 1 to B/null or null cells caused a dose-dependent decline in CFU-GM cloning efficiency. The addition of unstimulated autologous T cells to B/null or null cells enhanced CFU-GM growth both in the absence and presence of TGF-beta 1. T cells enhanced growth of all CFU-GM colony types to equal extents, whereas TGF-beta 1 inhibition preferentially reduced the proportion of macrophage colonies. TGF-beta 1 concentrations that blocked T-cell proliferation did not interfere with the capacity of T cells to enhance CFU-GM growth. The data suggest that noncycling T cells can reduce the inhibitory effects of TGF-beta 1, most likely through production of one or more stimulatory cytokines.

Cell Division↗

PubMed1990

Purification of peripheral blood hematopoietic cells using Campath-1M monoclonal antibody.

The purpose of this study was to develop a method for exhaustive depletion of T cells in preparation for allogeneic peripheral blood (PB) hematopoietic stem cell (HSC) transplantation. Our goal was to achieve 4 logs of T-cell reduction while minimizing the loss of colony-forming progenitor cells. Campath-1M, a rat IgM cytolytic to T cells, B cells, and monocytes in the presence of human complement, was chosen for the investigation. To determine the best target population for Campath treatment, three different PB mononuclear cell (MNC) populations were examined: 1) total MNC isolated with Ficoll-sodium diatrizoate; 2) MNC after T-cell depletion by sheep red blood cell rosetting and high capacity Percoll density gradient fractionation (E-MNC); and 3) B/Null cells, a population of E-MNC obtained by lysing monocytes with L-phenylalanine methyl ester (PME). Of the three target MNC populations, Campath-treated B/Null cells had the highest cloning efficiencies (CE) for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFUe) (56.7 and 38.5 colonies per 5 x 10(3) cells, respectively) and the highest enrichments for CFU-GM (108.2-fold, range 18- to 440-fold) and BFUe (178.5-fold, range 78- to 314-fold). Recoveries of progenitor cells were comparable for Campath-treated MNC (49.7% and 84.5%, respectively, for CFU-GM and BFUe) and Campath-treated B/Null (44.8% and 76.1%), and significantly higher than those from Campath-treated E-MNC (19.5% and 34.3%). The T-cell content in the Campath-treated B/Null cells were reduced by 5.0 logs from the starting MNC, as determined by limiting dilution analysis (LDA). We conclude that Campath-1M can be used for T-cell depletion of PB HSC. The best results were obtained when T cells and monocytes were removed prior to Campath treatment. This cell population is highly enriched for hematopoietic cells and may prove useful for in vitro studies as well allogeneic transplantation.

Antibodies, Monoclonal↗

PubMed1989

Removal of peripheral blood monocytes by phenylalanine methyl ester has no effect on the colony growth of hematopoietic progenitor cells.

Purification of hematopoietic cells often includes an adherence step for the removal of monocytes. Recent studies have shown that some hematopoietic cells are adherent and that plastic adhesion might activate monocyte/macrophage for cytokine production. We investigated the possibility of using L-phenylalanine methyl ester (PME) as an alternative approach to monocyte removal, particularly for large-scale cell separations. Cell yield, extent of monocyte removal and cloning efficiency of colony-forming progenitor cells were compared to that produced by plastic adhesion. Starting with mononuclear cells (MNC) from adult peripheral blood, cell recovery after PME treatment was higher than that achieved with adherence and fewer monocytes were present. Cloning efficiency of the granulocyte-monocyte colony-forming progenitor cells (CFU-gm) was not affected by the PME treatment. PME treatment and plastic adhesion of low-density cord blood cells produced comparable cell recovery, monocyte depletion and cloning efficiencies for CFU-gm and burst forming units for erythroid progenitor cells (BFU-e). The PME procedure was optimized for the purification of hematopoietic cells from large quantities (less than 1 x 10(9)) of low density. T cell-depleted peripheral blood MNC containing 60% to 85% monocytes. Compared to two cycles of plastic adhesion, PME treatment permitted higher cell recovery (5.9% vs. 1.3%), lower monocyte contamination (4.5% vs. 10.7%) and substantial cost, time and labor savings without compromising CFU-gm growth.

Cell Adhesion↗

PubMed1989

Allogeneic transplantation of blood-derived, T cell-depleted hemopoietic stem cells after myeloablative treatment in a patient with acute lymphoblastic leukemia.

This report describes an allogeneic peripheral blood stem cell transplant in a patient who had received marrow ablative therapy. The patient was an 18-year-old white male with acute lymphocytic leukemia in third remission for whom an allogeneic bone marrow transplant was recommended. His HLA-identical sibling preferred to donate peripheral blood stem cells rather than marrow. The donor cells were collected with 10 apheresis procedures and depleted of T lymphocytes to prevent excessive graft-versus-host disease. Nine collections were cryopreserved. The patient received high-dose cytosine arabinoside and 12 Gy of total body irradiation, followed by infusion of all cryopreserved donor cells. A portion of the tenth apheresis product collected on the day of transplant containing 1.8 x 10(9) T lymphocytes was infused without further processing to approximate the number of T lymphocytes given in an allogeneic bone marrow transplant; the remainder was T lymphocyte depleted and infused. More than 1 x 10(9)/l granulocytes were present on day +11. A bone marrow biopsy on day +27 showed trilineage engraftment. Cytogenetic studies demonstrated that the recipient's marrow and peripheral blood were populated exclusively with donor cells. Allogeneic peripheral stem cell transplantation produced an early hematopoietic engraftment. Since the patient died on day +32, sustained engraftment could not be evaluated.

Adolescent↗

PubMed1988

Granulocyte-monocyte progenitor cells from human peripheral blood: modulation of growth in vitro by T lymphocytes and monocytes.

Cell fractionation techniques have been used for the purification and characterization of hematopoietic cells present in peripheral blood and bone marrow. Following fractionation, the distribution of hematopoietic cells is frequently determined by the granulocyte-monocyte colony-forming unit (CFU-gm) assay. In this study, we questioned whether the purification process itself altered the sensitivity of the CFU-gm assay through changes in the accessory cell populations. Experiments showed that following T cell depletion, the cloning efficiency of CFU-gm was suboptimal, since the addition of autologous T lymphocytes was stimulatory, even when the concentration of conditioned medium was optimal. In contrast, growth of CFU-gm was inhibited by monocytes, both in the presence and absence of T cells. Sensitivity to monocyte-derived inhibition occurred at significantly lower monocyte concentrations when T cells were present. Thus, stem cell purification techniques which deplete T lymphocytes or enrich monocytes have an adverse effect on the cloning efficiency of peripheral blood CFU-gm. This study presents culture techniques capable of circumventing these problems.

Bone Marrow Cells↗

PubMed1987

Density distribution of hematopoietic progenitor cells and T lymphocytes from peripheral blood: heterogeneity of the human population.

Density gradient centrifugation has been used in animal systems to purify stem cells and eliminate T lymphocytes prior to allogeneic transplantation. There is substantial disagreement whether the same approach can be used to purify hematopoietic stem cells obtained from human peripheral blood. The purpose of the present study was to resolve that issue by determining the density distribution of 4 classes of human leucocytes: total mononuclear cells, T lymphocytes, CFUc, and BFUe. To ensure a representative sampling, a large number of randomly selected donors were analyzed. The results show that most T lymphocytes band between 1.068 and 1.071 g/ml, with relatively little variation from individual to individual. In contrast, the density distributions of both CFUc and BFUe fluctuated markedly from donor to donor. As a consequence, there was significant variability in the degree of progenitor-T cell separation. The implications of these results for clinical application of the density separation technique are discussed.

Cell Separation↗

PubMed1987

A new density gradient for the separation of large quantities of rosette-positive and rosette-negative cells.

Allogeneic transplantation of peripheral blood stem cells will not be feasible until new techniques are developed for large-scale depletion of T-lymphocytes. Small quantities of cells can be depleted of T-lymphocytes by sheep erythrocyte rosetting and Ficoll-diatrizoate discontinuous gradient fractionation. However, when processing 10(9)-10(10) cells, the fractionation step is inefficient because of the limited capacity of Ficoll-diatrizoate. We therefore developed a new discontinuous gradient for the separation of large numbers of sheep red blood cell (RBC) rosette-positive and negative cells. The gradient was designed so that sheep cells and rosettes would not interfere with the banding of rosette-negative cells. In that way, nonspecific entrapment was reduced, and high cell capacity and yield were achieved. The system fractionated rosetted cell suspensions into four populations: free sheep RBC, rosetted T cells, nonrosetted T cells, and low-density non-T cells. The 15-ml gradient routinely separated 3-24 X 10(8) cells. Progenitor yield ranged from 83% to 99%, with 95% depletion of T-lymphocytes. The method is rapid, reproducible, and inexpensive. This preparative technique could prove useful clinically when large-scale separation of E-rosette-positive and negative cells is required.

Cell Separation↗