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PubMed1998

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fc epsilon-RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways.

Cell Culture Techniques↗
PubMed1998

Morphological alterations in rat peritoneal mast cells by stem cell factor.

Stem cell factor (SCF) stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix. We found that the morphology of rat peritoneal mast cells (RPMC) altered after the addition of recombinant murine SCF (rmSCF) in vitro. The ability of rmSCF to enhance morphological alteration was dose dependent and completely abolished by anti-c-kit ACK2 monoclonal antibody. Exposure of RPMC to transforming growth factor-beta 1, wortmannin, genistein, herbimycin A, staurosporine, indomethacin and cytochalasin D before the addition of rmSCF antagonized rmSCF-induced morphological alteration. However, nordihydroguiaretic acid had no effect. Many RPMC appeared to respond also to nerve growth factor (NGF) but the total number of cells with altered morphology was much greater when the culture was stimulated by rmSCF than by NGF. We suggest that morphological alterations of mast cells by rmSCF is an important step for the participation in adhesion to tissue under resident physiological conditions.

Animals↗
PubMed2005

Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells.

Stem cell factor (SCF), which is well known as a cytokine capable of amplifying development and functions of mast cells, is mainly released from fibroblasts in the peripheral tissue. To investigate whether SCF controlled chemotactic migration of mast cells induced by IgE-specific Ag, murine bone marrow-derived cultured mast cells (BMCMC) and human cord blood-derived cultured mast cells (HuCMC) were preincubated with SCF. Although BMCMC and HuCMC sensitized with IgE directly moved toward specific Ag, preincubation for even 1 h with an optimal dose of SCF suppressed the IgE-mediated chemotactic movement. No or little inhibitory effect of SCF was detected in BMCMC derived from c-kit receptor-defect WBB6F1-W/Wv mice. In contrast, preincubation of BMCMC and HuCMC with SCF enhanced beta-hexosaminidase release and Ca2+ mobilization in response to Ag after sensitization with IgE. Using the real-time record of chemotactic migration, BMCMC preincubated with SCF manifested motionless without degranulation. These results suggest that locally produced SCF may have an inhibitory effect on chemotaxis of mast cells, contributing to their accumulation and enhancement of functions at the peripheral site in allergic and nonallergic conditions.

Animals↗
PubMed2000

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia: enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH(3).

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37 degrees C with 1 x 10(-9) mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 x 10(6) normal bone marrow cells and 5 x 10(4) leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl-lysophospholipid ET-18-OCH(3). An incubation for 4 h at 37 degrees C with 25 microg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET-18-OCH(3) with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.

Acute Disease↗
PubMed2002

Fibronectin combined with stem cell factor plays an important role in melanocyte proliferation, differentiation and migration in cultured mouse neural crest cells.

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild-type mice, we previously demonstrated that KIT-positive and/or L-3, 4-dihydroxyphenylalanine (DOPA)-positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT-positive cells and DOPA-positive cells were detected in the presence of SCF on ECM-coated wells than on non-coated wells. A statistically significant increase in DOPA-positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA-positive cells and KIT-positive cells were detected on either the ECM-coated or non-coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA-positive cells decreased with RGDS concentration in a dose-dependent fashion. Immunohistochemical staining revealed the presence of integrin alpha5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.

Animals↗
PubMed2000

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Animals↗
PubMed1999

Lyn is activated during late G1 of stem-cell-factor-induced cell cycle progression in haemopoietic cells.

Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical in haemopoiesis. Recently we found that the Src family member Lyn is highly expressed in SCF-responsive cells, associates with c-Kit and is activated within minutes of the addition of SCF. Here we show that SCF activates Lyn a second time, hours later, during SCF-induced cell cycle progression. In cells arrested at specific phases of the cell cycle with the drugs mimosine, aphidicolin and nocodazole, maximal Lyn kinase activity occurred in late G(1) and through the G(1)/S transition. Similarly, kinetic studies of SCF-induced cell cycle progression found that activation of Lyn preceded the G(1)/S transition and was maintained into early S-phase. Activation of Lyn was paralleled by two events critical for the G(1)/S transition, increases in cyclin-dependent kinase 2 (Cdk2) activity and phosphorylation of the retinoblastoma gene product (Rb). Lyn was associated with Cdk2; Cdk2-associated Lyn was heavily phosphorylated on serine and threonine residues both in vitro and in situ during S-phase. Inhibition of Lyn activity with PP1 disrupted association with Cdk2 and decreased the numbers of cells entering S-phase. The degree of phosphorylation of Rb in PP1-treated cells suggested an increased number of cells arrested in the middle of G(1). These findings demonstrate that SCF activates the Src family member Lyn before the G(1)/S transition of the cell cycle and suggest that Lyn is involved in SCF-induced cell cycle progression.

Aphidicolin↗
PubMed1993

Expression of beta 1 tubulin (beta Tub56D) in Drosophila testis stem cells is regulated by a short upstream sequence while intron elements guide expression in somatic cells.

Stem cell differentiation to mature spermatozoa is a morphogenetic process that is highly dependent on microtubular arrays. In the early, mitotically active stages of spermatogenesis, only the beta 1 tubulin isotype is expressed. Analysis of transgenic flies containing beta 1-lacZ gene fusions revealed that this expression is regulated by sequences located between positions -45 and -191 upstream of the transcription initiation site. Furthermore, beta 1 tubulin is a major component of cyst cells. Expression in these cells is driven by enhancer elements located in the beta 1 tubulin gene intron. These enhancer elements also guide expression in combination with the hsp70 basal promoter. In addition, redundant enhancer elements in the intron drive expression in the testis wall. Our data show that within a single tissue, the male gonad, expression of the beta 1 tubulin gene is under cell-type-specific control mediated by independent cis-acting elements. Therefore in the germ line, control of beta 1 tubulin expression is strictly governed by promoter-proximal elements, while for the somatic parts of the testis, enhancer elements confer less stringent expression control.

Animals↗
PubMed1996

A possible change in doubling time of haemopoietic progenitor cells with stem cell development.

We separated haemopoietic progenitors derived from marrow cells of 5-fluorouracil (5-FU)-treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum-free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5-FU-treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin-3 (IL-3), IL-11. and steel factor (SF), Cell-cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.

Animals↗
PubMed2006

Why do chronic myelogenous leukemia stem cells survive allogeneic stem cell transplantation or imatinib: does it really matter?

It is generally accepted that allogeneic stem cell transplantation can 'cure' chronic myelogenous leukemia (CML), although occasional patients relapse more than 10 years after the transplant procedure. Such cures presumably result from the combined effects of leukemia stem cells (LSCs) of the conditioning regimen and the graft-vs.-leukemia (GvL) effect mediated by donor-derived T lymphocytes. The advent of imatinib has revolutionized the management of patients with CML, but much evidence suggests that it does not eradicate all LSCs, which theoretically remain a potential source of relapse to chronic phase or advanced phase disease. Moreover, sub-clones of Philadelphia-positive cells bearing mutations that code for amino-acid substitutions in the Bcr-Abl kinase domain can be identified in patients receiving treatment with imatinib and are associated with varying degrees of resistance to this agent. In the present review, we postulate that LSCs, similar to their normal counterparts, may alternate between cycling and quiescent modes. In the cycling mode, they may express Bcr-Abl protein and be susceptible to the acquisition of additional mutations, whereas, in the quiescent mode, they may express little or no Bcr-Abl oncoprotein, cannot acquire additional mutations and are unaffected by imatinib. Thus, a patient who starts treatment early in the natural history of CML, and who responds to imatinib clinically, may not have had the opportunity to acquire additional mutations in LSCs. In this case, the persistence long-term of quiescent 'non-mutated' LSCs despite imatinib treatment might be consistent with freedom from relapse to chronic or advanced phase disease, provided that they remain vulnerable to imatinib when they are recruited into cycle. Conversely, when imatinib resistant Philadelphia-positive sub-clones predominate, this is likely to be due to the recruitment to hematopoiesis of quiescent stem cells that had been in cycle before administration of imatinib and that had acquired additional mutations; in such cases, the best approach to eradication of residual LSCs might be to target expressed proteins thought to be targets for the GvL effect.

Benzamides↗
PubMed2005

Molecular mechanisms of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics.

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose-response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose-response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis.

Breast↗
PubMed2000

CD34+ selected cells in mismatched stem cell transplantation: a single centre experience of haploidentical peripheral blood stem cell transplantation.

Over the past 3 years we have performed 10 haploidentical peripheral blood stem cell transplants in patients with incurable haematological malignancies and no prospect of a matched unrelated donor within an adequate time period. Conditioning consisted of ATG, TBI, thiotepa, cyclophosphamide and additional radioimmunotherapy in five patients. All patients received G-CSF mobilized peripheral blood stem cell grafts. GVHD prophylaxis consisted of T cell depletion by CD34+ selection; no post-transplant immunosuppression was given in nine patients. Stable engraftment was achieved in nine patients; one case of acute graft rejection was observed. Seven patients developed grade I acute GVHD, and six patients have developed chronic GVHD. Infections were the most significant clinical problem post transplant. Two patients have suffered a relapse of their disease and two further patients have died of transplant-related complications. After a median follow-up of 13 months (range 5-37 months) six patients are surviving in remission. We conclude that haploidentical PBSCT is a reasonable alternative to a MUD transplant.

Adult↗
PubMed2001

Flow cytometric assessment of lymphocyte subsets, lymphoid progenitors, and hematopoietic stem cells in allogeneic stem cell grafts.

Currently, bone marrow (BM), cord blood (CB), and G-CSF-mobilized peripheral blood progenitor cells (PBPCs) are the most commonly used sources for allogeneic stem cell transplantation (SCT). The aim of this study was to assess the yields and distribution of lymphocyte subsets, lymphocyte progenitors and hematopoietic stem cells (HSC) in each type of allograft by three-color flow cytometry. The yields of CD34(+)CD38(-) HSCs did not differ significantly between BM grafts (2.80 +/- 0.74 x 10(6)) and leukapheresis products (LPs) (1.82 +/- 0.64 x 10(6)), and were lowest in CB grafts (0.21 +/- 0.05 x 10(6)). For most lymphocyte subsets yields were lowest in CB grafts and significantly higher in LPs than in BM grafts. BM grafts, however, contained the highest yields of CD34(+)CD19(+)CD20(-) B cell progenitors and CD19(+)CD20(-) B cells. The relative frequencies of the naive CD45RA(+)CD45RO(-) phenotype among CD4(+) and CD8(high) T cells were highest in CB grafts (P < or = 0.001), and higher in LPs than in BM grafts (P < or = 0.02). The latter finding was in accordance with a preferential G-CSF mobilization of naive T cells relative to the total lymphocyte population (P < or = 0.014). CD3(+)CD8(low) and CD3(+)CD8(low)CD4(-) subsets, which facilitate engraftment in murine transplantation models, demonstrated a tendency towards lower frequencies among T cells in CB grafts and LPs compared to BM grafts. This observation coincided with a significantly reduced mobilization of subsets potentially enriched for facilitating cells as compared to the total lymphocyte population (P < or = 0.036). The CD34(+) compartment of CB grafts contained a significantly higher percentage (12.1%) of CD34(+)CD7(+)CD3(-) T cell progenitors than those of BM grafts (5.1%) and LPs (3.6%). In addition, CB lymphocytes contained the highest fraction of CD3(-)CD16/56(+) NK cells (P < or = 0.013) and almost no CD3(+)CD16/56(+) NKT cells (P < 0.001) compared to adult cell sources. In summary, LPs, CB allografts and BM allografts differ widely with respect to the cellular composition of their lymphocyte compartments, which is partially affected by a varying mobilization efficiency of G-CSF for distinct lymphocyte subsets.

Adult↗
PubMed2001

Dedifferentiation of epidermal cells to stem cells in vivo.

The effects of growth factors on wound healing have been studied extensively; however epidermal regeneration is not fully understood. We treated eight patients with leg ulcers with recombinant human epidermal growth factor (rhEGF) and compared biopsies of regenerating epidermis with those of controls who did not receive rhEGF. We used immunohistochemistry to identify cells expressing keratin 19 and beta1 integrin in regenerated epidermis from patients and controls. Patients treated with rhEGF had stem cells in the spinous and granular layers of regenerated epidermis. Histological analysis showed that these stem cells had reverted from differentiated to undifferentiated stem cells. Our findings provide evidence for epidermal cell reversion.

Adult↗
PubMed2000

Serum-soluble c-kit levels during mobilization of peripheral blood stem cells correlate with stem cell yield.

c-Kit is expressed in hematopoietic stem cells and plays an important role in hematopoiesis. In 16 patients with malignancies, serum-soluble c-Kit levels and the expressions of c-KIT messenger RNA (mRNA) and protein in peripheral blood mononuclear cells were analyzed serially during 26 courses of peripheral blood stem cell (PBSC) mobilization after granulocyte colony-stimulating factor administration following chemotherapy for PBSC harvest. Serum-soluble c-Kit levels were significantly lower in patients than in controls (179.7+/-17.7 arbitrary units [AU]/mL versus 274.5+/-18.9 AU/mL; P < .001), decreasing after chemotherapy (167.7+/-18.2 AU/mL), increasing from day 14, and peaking at day 19 (193.3+/-16.4 AU/mL). The numbers of both c-Kit+ cells and CD34+ cells and granulocyte-macrophage colony-forming units in peripheral blood peaked at day 17, following the peak of the expression of c-KIT mRNA. Serum-soluble c-Kit levels showed a significant positive correlation with the numbers of CD34+ cells in both peripheral blood and leukapheresis products (r = 0.553, P < .01, and r = 0.640, P < .001, respectively) and changed at higher levels in patients with large numbers of PBSCs versus patients with small numbers of PBSCs (P < .05). Serum-soluble c-Kit may reflect the capacity for hematopoiesis after chemotherapy and may be useful in predicting the number of PBSCs that can be mobilized and harvested after mobilization, as well as for monitoring the timing for PBSC harvest.

Adult↗
PubMed2006

Embryonic germ cells: when germ cells become stem cells.

Embryonic germ cells (EGCs) are pluripotent stem cells derived from primordial germ cells (PGCs). PGCs are progenitors of adult gametes, which diverge from the somatic lineage between late embryonic to early fetal development. First derived in the mouse, EGCs have also been derived from human, chicken, and pig. As pluripotent stem cells, EGCs demonstrate long-term self-renewal via clonal expansion in an undifferentiated state, and differentiate in vitro to form embryoid bodies containing cells that represent all three germ layers as well as mixed cell populations of less differentiated progenitors and precursors. This is also demonstrated in vivo by their formation into experimentally induced teratocarcinomas following transplantation. Furthermore, mice, pig, and chicken EGCs have also been shown to contribute to experimentally produced chimeric animals, including germline transmission. Importantly, EGCs demonstrate normal and stable karyotypes as well as normal patterns of genomic imprinting, including X-inactivation. Transplantation studies have begun in a variety of models in hopes of defining their potential use to treat a wide variety of human conditions, including diabetes and urological and neurological disorders.

Animals↗
PubMed2002

Kinetics of stem cell engraftment and clearance of leukaemia cells after allogeneic stem cell transplantation with reduced intensity conditioning in chronic myeloid leukaemia.

It is hypothesised that an effective graft-vs.-leukaemia reaction contributes substantially to the therapeutic effect of reduced intensity conditioning stem cell transplantation in chronic myeloid leukaemia. However, kinetic data on eradication of leukaemia cells and stem cell engraftement which could support this assumption are lacking. Thus, we investigated bcr/abl fusion transcripts and haematopoietic chimerism in 14 patients undergoing such a transplantation protocol. Ten of them obtained a complete molecular remission, and two patients achieved haematologic remissions but remained bcr/abl positive. Weekly determinations of bcr/abl transcript numbers by qualitative and quantitative polymerase chain reaction and donor chimerism revealed that 10 responders cleared bcr/abl positive cells from the peripheral blood within a median of 9 wk (range 3-22 wk). The close relation (P = 0.0075) between the first occurrence of graft-vs.-host disease and the complete clearance of bcr/abl positive blood cells argues in favour of an effective graft-vs.-leukaemia reaction.

Adult↗
PubMed2003

When is a stem cell really a stem cell?

While bone marrow transplantation has long been established as an effective approach to the clinical management of a variety of malignant and nonmalignant diseases, the future application of pluripotent stem cells in transplant settings promises to deliver this therapy to a much broader range of indications. In this review, I summarize the emerging field of embryonic stem cell biology in the context of potential clinical applications and regulatory issues.

Animals↗