Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant.
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We investigated bone marrow (BM) and circulating (PB) hematopoietic progenitor cells in 37 normal donors and in 25 patients 1 to 8 years after successful allogeneic bone marrow transplant. At the time of testing, transplanted patients had normal blood counts and bone marrow cellularity. By flow cytometry, BM CD34+ cells were found to be three- to four-fold decreased in transplanted patients compared to normal donors, while the number of PB CD34+ cells was the same as in normal donors. Using a methylcellulose colony assay, primary BM colony-forming cells (CFU-GM) were decreased 2.1-fold, whereas PB CFU-GM were only marginally decreased. In a long-term culture initiating cell (LTC-IC) assay, an eight-fold decrease of early progenitor cells was observed in the marrow of transplanted patients compared to normal donors, and a five-fold decrease was documented in peripheral blood. We found that the BM LTC-IC cell number correlated with concurrently determined BM CD34+ cells and committed progenitor cell number (measured as CFU-GM) and with PB LTC-IC number, but not with PB CFU-GM and CD34+ cells. We conclude that marrow and circulating early stem cell compartments, as measured by the LTC-IC assay, are greatly and permanently depressed following bone marrow transplant. The correlation between BM and PB LTC-IC indicates that the enumeration of circulating LTC-IC can be used as a measure of the stem cell compartment in the bone marrow after transplant. It seems that the deficiency of the most immature progenitor cells persists forever after successful bone marrow transplant; this means that a complete hematopoietic reconstitution can be sustained by a reduced stem cell pool. (+info)
Counterflow centrifugation allows addition of appropriate numbers of T cells to allogeneic marrow and blood stem cell grafts to prevent severe GVHD without substantial loss of mature and immature progenitor cells.
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Using counterflow centrifugation elutriation (CCE) lymphocytes can be separated from CD34+ populations based on size. Immature progenitors tend to be smaller than mature cells suggesting that CCE introduces loss of stem cells. We compared the separation of 12 PBSC with 16 BM transplants. Cells were separated in 12 fractions (3000-2200 r.p.m.) and the rotor off (RO) fraction. Separation patterns of BM and PBSC were comparable. B cells were collected in the high speed fractions followed by T and NK cells. In contrast, progenitor cells were collected in lower speed fractions. By adding successively T cell-depleted fractions to the RO fraction a BM transplant could be composed containing 0.7 x 10(6) T cells/kg and 90%, 89% and 68% recovery of CD34+, CFU-GM and BFU-E. PBSC were separated in four CCE runs inducing higher numbers of T cells in the graft (4.4 x 10(6)/kg) and 54% CD34+, 46% CFU-GM and 37% BFU-E recovery. Time of engraftment was not delayed and no graft failure was observed. The higher number of T cells was not associated with higher incidence of GVHD. Acute GVHD > or = grade III occurred in 0 of 16 BM and two of 12 PBSC recipients; extensive chronic GVHD was observed in four of 15 and three of nine recipients, respectively. To study immature cells in the graft, CD34 subpopulations and cells with long-term repopulating ability, determined using cobble-stone area formation (CAFC assay), were evaluated in each fraction. The separation patterns in BM and PBSC were comparable. Cells with mature and immature phenotype were enriched in lower speed fractions (mean recovery of 74% CD34+/CD13-/DR-). The CAFC week 2, 4 and 6 were also enriched in these fractions. These data show that the used CCE procedure is a reliable method to deplete T cells from stem cell transplants without substantial loss of immature and mature progenitors. (+info)
A preliminary study of ultrasound aspiration of bone erosion in early rheumatoid arthritis.
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OBJECTIVE: To develop a new technique to assess the primary lesion in early rheumatoid arthritis (RA). METHODS: Ten patients with early RA and radiographically or MRI confirmed erosions had a needle introduced into the base of the erosion under sonographic guidance. Material was then aspirated from this site. RESULTS: The procedure was well tolerated with no complications. Small samples of necrotic bone and tissue were obtained in five out of 10 cases. In one case, a distinctive population of pleomorphic CD34 + cells with characteristics of bone marrow progenitors was isolated. Tissue invading bone with a characteristic appearance of pannus was not seen. CONCLUSION: A new method of sampling the earliest lesion in RA is described. The findings raise questions about the nature of bone damage in early RA. (+info)
Human CD34(+) cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus.
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Human CD34(+) hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34(+) cells, although it was expressed at lower density on MPB with respect to BM CD34(+) cells. Freshly isolated and in vitro-cultured CD34(+) cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Of interest, CD34(+)/CD38(+) committed progenitor cells, unlike primitive CD34(+)/CD38(-) cells, expressed SDF-1 mRNA. Supernatants from in vitro-cultured CD34(+) cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34(+) cells. Because CD34(+) cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34(+) cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34(+) cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4(+) T cells or CD34(+) cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34(+) cells. However, CXCR4 on CD34(+) cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34(+) progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34(+) cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4. (+info)
Ex vivo expansion of autologous bone marrow CD34(+) cells with porcine microvascular endothelial cells results in a graft capable of rescuing lethally irradiated baboons.
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Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host. (+info)
Differential expression of KDR/VEGFR-2 and CD34 during mesoderm development of the early human embryo.
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Recent findings on vertebrate embryos have provided compelling evidence for the existence of hemangioblasts, i.e. common precursors for endothelial and hematopoietic cells, characterized by expression of the VEGFR2/Flk1 receptor. We describe here a population of KDR+ CD34- mesoderm cells that emerges in early-somitic human embryos, by the beginning of the 4th week of gestation. In the developing blood vessels, KDR-expressing CD34- cells gradually coexpress increasing levels of CD34 antigen. Remarkably, as development proceeds, a KDR+ CD34- contingent persists in the paraaortic splanchnopleura until just prior to the emergence of aorta-associated hematopoietic cell clusters. These observations suggest that KDR+ CD34- mesodermal cells might represent the putative hemangioblastic precursor of human hematopoietic and endothelial lineages. (+info)
Culturing human umbilical cord blood: a comparison of mononuclear vs CD34+ selected cells.
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We compared UCB mononuclear cells (MNC) with CD34+ selected cells in a serum-free static culture system. Cell number proliferation of MNCs was inferior to CD34+ selected cells. MNCs, however, showed a substantial increase from 0.94% CD34+ cells on day 0 to 5.8% on day 7, whereas in the CD34+ selected samples the CD34+ cell content declined continously from 62.2% on day 0 to 27.7% on day 7. The number of CFU-GM increased during culture of both cell fractions. Here, only the MNCs showed a substantial increase in clonogenicity on day 7 and day 14 to 11.1- and 4.1-fold input, respectively. This expansion of the CD34+ progenitor cell pool in the MNCs fraction was at least in part attributable to T cells, since the physical abrogation of T cells blocked this effect. Refeeding and reseeding of cells on day 7 had stimulating effects especially on the CD34+ cells, where cell number proliferation increased from 16.3-fold without to 58.1-fold on day 14. Also, we could find sporadic chromosomal aberrations in four of 100 metaphases examined after 7-20 days of ex vivo expansion. The significance of this observation needs to be clarified in a larger series. (+info)
Using at least 5x10(6)/kg CD34+ cells for autologous stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation.
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For autologous stem cell transplantation, it is common practice to infuse at least 2 x 10(6)/kg CD34+ cells to ensure rapid engraftment. However it was recently claimed that increasing the threshold to 5 x 10(6)/kg leads to a faster platelet engraftment. To evaluate these threshold values in our patient population we undertook a retrospective analysis of 127 autologous transplants performed at our institution between 1992 and 1998. Diagnoses included Hodgkin's and non-Hodgkin's lymphoma, myeloma, acute leukaemias and solid tumours. The transplant was peripheral blood stem cells in 107 cases and CD34-selected peripheral blood stem cells in 20 cases. The median number of transplanted CD34+ cells was 3.2 x 10(6)/kg (range 0.64-25.9 x 10(6)/kg). Haematopoietic recovery to a neutrophil count >0.5 x 10(9)/l took a median of 10 (range 5-16) days from transplant. When comparing patients receiving at least 5 x 10(6)/kg and 2-5 x 10(6)/kg CD34+ cells we found a significant reduction in the median number of days with fever (1 vs 3.5 days, P = 0.0025), incidence of fever (78.8 vs 92.1%, P = 0.032) as well as duration of antibiotic treatment (7 vs 10 days, P = 0.038). This was paralleled by a faster neutrophil recovery to 0.5 x 10(9)/l (9 vs 10 days, P = 0.047). There was no significant difference in the number of platelet or red cell transfusions between the two groups. We conclude that transplantation with a stem cell dose of at least 5 x 10(6)/kg CD34+ cells reduces infectious complications and should thereby increase the safety of this type of therapy while reducing duration (and cost) of antibiotic therapy. The transplantation threshold should thus not remain at 2 x 10(6)/kg particularly in patients with a good stem cell mobilisation capacity. (+info)