scholarly journals Phenotypic and functional characterization of long-term culture- initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2033-2042 ◽  
Author(s):  
F Prosper ◽  
D Stroncek ◽  
CM Verfaillie
Keyword(s):  
Steady State ◽  
Absolute Number ◽  
Granulocyte Colony Stimulating Factor ◽  
Hla Dr ◽  
Normal Individuals ◽  
Cd34 Cells ◽  
The Absolute ◽  
Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) have successfully been used as stem cells for both autologous and allogeneic transplants. However, little is known concerning the absolute number and phenotype of primitive progenitors, such as long-term culture-initiating cells (LTC-IC) in mobilized PBPC. The aim of our study was to evaluate the capacity of G- CSF to mobilize LTC-IC in the PB of normal individuals and to evaluate the phenotypic and functional characteristics of G-CSF mobilized LTC- IC. G-CSF was administered to 29 healthy volunteers at 7.5 micrograms or 10 micrograms/kg/d subcutaneously (SC) for 5 consecutive days and PBPC were harvested on day 6. Mobilization with G-CSF increased the absolute number of week 5 LTC-IC in PB 60-fold, while the number of CD34+ cells and committed colony forming cells (CFC) was increased sevenfold to 12-fold. The frequency of CFC and week 5 LTC-IC in CD34+ cells selected by fluorescence-activated cell sorter (FACS) from mobilized PBPC was 2 +/- 0.3-fold and 9 +/- 2.2-fold higher respectively than in CD34+ cells selected from unmobilized PBMNC. CFC were enriched in the CD34+ CD38+ and CD34+ HLA-DR+ populations. The absolute number of LTC-IC present in CD34+ CD38- and CD34+ HLA-DR- cells selected by FACS from either mobilized PBPC, unmobilized PBMNC or steady state bone marrow (BM) was similar (0.5% to 2%). In contrast to unmobilized PBMNC or steady state BM CD34+ CD38+ and CD34+ HLA-DR+ cells, which contain less than 0.1% LTC-IC, CD34+ CD38+ and CD34+ HLA- DR+ cells sorted from mobilized PBPC contained 0.5% to 5% of cells capable of sustaining hematopoiesis in long-term cultures for 5 weeks. However, 90% to 95% of LTC-IC present in mobilized CD34+ CD38+ and CD34+ HLA-DR+ cells were not able to sustain hematopoiesis for 8 weeks, while 30% of CD34+ CD38- and CD34+ HLA-DR- LTC-IC present in mobilized PBPC could sustain hematopoiesis for at least 8 weeks. This suggests that the majority of CD34+ CD38+ and CD34+ HLA-DR+ week 5 LTC-IC represent progenitors at an intermediate state of differentiation. We conclude that G-CSF effectively mobilizes LTC-IC in the blood of normal individuals. Although a fraction of these cells has functional characteristics similar to those of steady state PBMNC or BM LTC-IC, more than 85% of mobilized PBPC LTC-IC are CD34+ CD38+ and CD34+ HLA- DR+, capable of sustaining hematopoiesis for 5 weeks, but not for 8 weeks. The functional and phenotypic characterization of primitive and more mature populations of LTC-IC in mobilized PBPC should prove extremely useful in future studies examining the role of these progenitors in engraftment following transplantation.

scholarly journals Primitive Long-Term Culture Initiating Cells (LTC-ICs) in Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cells Have Similar Potential for Ex Vivo Expansion as Primitive LTC-ICs in Steady State Bone Marrow

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3991-3997 ◽  
Author(s):  
Felipe Prosper ◽  
Kirk Vanoverbeke ◽  
David Stroncek ◽  
Catherine M. Verfaillie
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hla Dr ◽  
Stimulating Factor

Abstract We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+HLA-DR− and CD34+CD38− and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+HLA-DR+ cells (rich in mature LTC-IC) and CD34+HLA-DR− cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF ) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1α) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+HLA-DR− mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+HLA-DR− PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1α were added to noncontact cultures (96 ± 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of “mature” CD34+HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+HLA-DR− LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1α for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.

scholarly journals Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4523-4531 ◽  
Author(s):  
Frederick D. Goldman ◽  
Geraldine Aubert ◽  
Al J. Klingelhutz ◽  
Mark Hills ◽  
Sarah R. Cooper ◽  
...  
Keyword(s):  
Dyskeratosis Congenita ◽  
Normal Numbers ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Normal Differentiation ◽  
Stimulating Factor ◽  
Mobilized Peripheral Blood

Abstract Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34+ cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34+ subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34+ DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.

scholarly journals Blood-derived autografts collected during granulocyte colony- stimulating factor-enhanced recovery are enriched with early Thy-1+ hematopoietic progenitor cells

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1936-1943 ◽  
Author(s):  
R Haas ◽  
R Mohle ◽  
M Pforsich ◽  
S Fruehauf ◽  
B Witt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mean Fluorescence Intensity ◽  
Progenitor Cell ◽  
Enhanced Recovery ◽  
Granulocyte Colony Stimulating Factor ◽  
Hematopoietic Progenitor ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Stimulating Factor

It was the objective of the study to characterize CD34+ hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) in a group of 24 cancer patients. After cytotoxic chemotherapy, R-metHu granulocyte colony-stimulating factor (R-metHuG-CSF; filgrastim, 300 micrograms daily, subcutaneously) was given to shorten the time of neutropenia as well as to increase the rebound of peripheral blood progenitor cells (PBPC) for harvesting. The proportion of CD34+ cells in the leukapheresis products (LPs) was 1.4-fold greater than in BM samples that were obtained at the same day (LP: median, 1.4% v BM: median, 1.0%, P < .01). Two- and three-color immunofluorescence showed that blood-derived CD34+ cells comprised a greater proportion of a particular early progenitor cell than CD34+ cells of bone marrow. Blood-derived progenitor cells tended to have a higher mean fluorescence intensity of CD34 and expressed significantly lower levels of HLA-DR (mean fluorescence intensity of HLA-DR: 442.6 +/- 44.9 [LP] v 661.5 +/- 64.6 [BM], mean +/- SEM, P < .01). Furthermore, the blood-derived CD34+ cells comprised a 1.7-fold greater proportion of Thy-1+ cells (LP: median, 24.4% v BM: median, 14.4%, P < .001) and expressed significantly less c-kit (LP: median, 20.5% v BM: median, 31.0%, P < .01). Three-color analysis showed that high levels of Thy-1 expression were restricted to CD34+/HLA-DRdim or CD34+/HLA-DR-cells confirming the early developmental stage of this progenitor cell subset. The proportion of CD34+/CD45RA(bright) cells representing late colony-forming unit granulocyte-macrophage (CFU-GM) was smaller in LPs compared with BM (P < .05). For an examination of BM CD34+ cells before the mobilization chemotherapy, samples of 16 patients were available. The mean proportion of c-kit expressing CD34+ cells in the bone marrow during G-CSF-stimulated reconstitution decreased 1.8-fold compared with baseline values. There was no difference in the proportion of BM-derived CD34+/Thy-1+ cells and CD34+/CD45RA+ cells between steady-state hematopoiesis and G-CSF-supported recovery. Our data suggest that during G-CSF-enhanced recovery, CD34+ cells in the PB are enriched with more primitive progenitor cells to evenly replenish the BM after the chemotherapy-related cytotoxic damage.

scholarly journals Cycling Status of CD34+ Cells Mobilized Into Peripheral Blood of Healthy Donors by Recombinant Human Granulocyte Colony-Stimulating Factor

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1189-1196 ◽  
Author(s):  
Roberto M. Lemoli ◽  
Agostino Tafuri ◽  
Alessandra Fortuna ◽  
Maria Teresa Petrucci ◽  
Maria Rosaria Ricciardi ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Significant Proportion ◽  
S Phase ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Stimulating Factor

Abstract In this study, we assessed the functional and kinetic characteristics of highly purified hematopoietic CD34+ cells from the apheresis products of 16 normal donors undergoing glycosylated granulocyte colony-stimulating factor (G-CSF ) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation in allogeneic recipients. Mobilized CD34+ cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) CSF in vitro and the content of very primitive long-term culture initiating cells (LTC-IC). In addition, the cycling status of circulating CD34+ cells, including committed clonogenic progenitor cells and the more immature LTC-IC, was determined by the cytosine arabinoside (Ara-C) suicide test and the acridine orange flow cytometric technique. By comparison, bone marrow (BM) CD34+ cells from the same individuals were studied under steady-state conditions and during G-CSF administration. Clonogenic assays in methylcellulose showed the same frequency of colony-forming unit cells (CFU-C) when PB-primed CD34+ cells and BM cells were stimulated with phytohemagglutinin–lymphocyte-conditioned medium (PHA-LCM). However, mobilized CD34+ cells were significantly more responsive than their steady-state BM counterparts to interleukin-3 (IL-3) and stem cell factor (SCF ) combined with G-CSF or IL-3 in presence of erythropoietin (Epo). In cultures added with SCF, IL-3, and Epo, we found a mean increase of 1.5- ± 1-fold (standard error of the mean [SEM]) of PB CFU–granulocyte-macrophage and erythroid progenitors (burst-forming units-erythroid) as compared with BM CD34+ cells (P < .05). Conversely, circulating and BM megakaryocyte precursors (CFU-megakaryocyte) showed the same clonogenic efficiency in response to IL-3, granulocyte-macrophage–CSF and IL-3, IL-6, and Epo. After 5 weeks of liquid culture supported by the engineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we reported 48.2 ± 35 (SEM) and 62.5 ± 54 (SEM) LTC-IC per 104 CD34+ cells in PB and steady-state BM, respectively (P = not significant). The Ara-C suicide assay showed that 4% ± 5% (standard deviation [SD]) of committed precursors and 1% ± 3% (SEM) of LTC-IC in PB are in S-phase as compared with 25.5% ± 12% (SD) and 21% ± 8% (SEM) of baseline BM, respectively (P < .001). However, longer incubation with Ara-C (16 to 18 hours), in the presence of SCF, IL-3 and G-CSF, or IL-6, showed that more than 60% of LTC-IC are actually cycling, with no difference being found with BM cells. Furthermore, studies of cell-cycle distribution on PB and BM CD34+ cells confirmed the low number of circulating progenitor cells in S- and G2M-phase, whereas simultaneous DNA/RNA analysis showed that the majority of PB CD34+ cells are not quiescent (ie, in G0-phase), being in G1-phase with a significant difference with baseline and G-CSF–treated BM (80% ± 5% [SEM] v 61.9% ± 6% [SEM] and 48% ± 4% [SEM], respectively; P < .05). Moreover, G-CSF administration prevented apoptosis in a small but significant proportion of mobilized CD34+ cells. Thus, our results indicate that mobilized and BM CD34+ cells can be considered equivalent for the frequency of both committed and more immature hematopoietic progenitor cells, although they show different kinetic and functional profiles. In contrast with previous reports, we found that PB CD34+ cells, including very primitive LTC-IC, are cycling and ready to progress into S-phase under CSF stimulation. This finding should be taken into account for a better understanding of PBSC transplantation.

scholarly journals Flt3 Ligand Enhances the Yield of Primitive Cells After Ex Vivo Cultivation of CD34+ CD38dim Cells and CD34+ CD38dim CD33dim HLA-DR+ Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3903-3913 ◽  
Author(s):  
Douglas C. Dooley ◽  
Mang Xiao ◽  
Barbara K. Oppenlander ◽  
J. Michael Plunkett ◽  
Stewart D. Lyman
Keyword(s):  
Ex Vivo ◽  
Absolute Number ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitors ◽  
Colony Forming Unit ◽  
Cell Pool ◽  
Hla Dr ◽  
Cd34 Cells ◽  
The Impact

Abstract 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 CD33dim 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+ CD33dim 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.

scholarly journals Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

1988 ◽  
Vol 256 (1) ◽  
pp. 213-218 ◽  
Author(s):  
P Wingfield ◽  
R Benedict ◽  
G Turcatti ◽  
B Allet ◽  
J J Mermod ◽  
...  
Keyword(s):  
Coli Strain ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Disulphide Bonds ◽  
Disulphide Bond Formation ◽  
Stimulating Factor ◽  
Factor G

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

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