Biomedical subjects

C M Verfaillie

Publications and source records attributed to C M Verfaillie.

At least 73 records · Page 4Linked to original sources

PubMed1997

A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium.

FACS-selected CD34+ HLA-DR- cells (DR- cells) may provide a source of benign stem cells suitable for autografting in chronic myelogenous leukemia (CML) and other hematological malignancies. However, DR- cell selection depletes the majority of committed hematopoietic progenitors, which may be important for early engraftment. Furthermore, only a small number of DR- cells may be selectable in certain patients. These impediments to the use of DR- cells for autografting may be overcome through the development of ex vivo culture systems that support expansion and initial differentiation of primitive progenitors. Because 2-week culture of DR- cells in a stroma "noncontact" system supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein 1-alpha (MIP-1alpha) expands both long-term culture-initiating cells (LTC-ICs) and colony-forming cells (CFCs), we adapted this system to a clinically applicable method for expanding LTC-ICs and CFCs ex vivo. In initial small-scale studies, DR cells were grown in stroma conditioned medium (SCM) supplemented with IL-3 with or without additional growth-promoting cytokines and the chemokines PF-4 and BB10010, all approved for clinical use. An IL-3 dose-dependent expansion of committed progenitors and LTC-ICs was observed when DR- cells were cultured in tissue culture plates in SCM+IL-3 for 2 weeks. Similar CFC expansion along with increased (5-fold) LTC-IC expansion was observed following addition of PF-4 to SCM+IL-3 cultures. The addition of stem cell factor (SCF), but not of IL-6, IL-11, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, IL-1, and IL-7, increased CFC and LTC-IC expansion beyond the levels observed with SCM+IL-3 alone. We next evaluated the suitability of this culture system for scale-up. Culture of 2-6 x 10(5) DR- cells in gas-permeable bags with SCM+IL-3 resulted in similar CFC and LTC-IC expansion as seen in small-scale cultures. In addition, we observed that progenitors capable of differentiating to natural killer (NK)-cells were maintained under these conditions. Finally, we found that BCR/ABL mRNA-negative CFCs and LTC-ICs present in DR- cells selected from steady-state CML marrow could be expanded in large-scale SCM+IL-3 cultures. We conclude that culture of DR- cells for 2 weeks in SCM+IL-3 culture, with or without PF-4 or SCF, results in significant CFC and LTC-IC expansion and lymphoid NK progenitor maintenance. This culture system is readily adaptable to the expansion of primitive progenitors for autotransplantation.

Bone Marrow Cells↗

PubMed1996

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.

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.

ADP-ribosyl Cyclase↗

PubMed1996

BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients.

We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) CML patients. Fluorescence-activated call-sorting (FACS)-selected 34+DR- and 34+DR+ cells were subjected to reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization. These cells were also cultured in long-term bone marrow culture for 1 to 5 weeks to examine the number of LTC-IC and the presence or absence of the BCR/ABL gene rearrangement in progeny of primitive LTC-IC. The number of 34+DR- cells and LTC-IC present in ECP CML marrow was similar to that in normal (NL) marrow, whereas the numbers were reduced in AP CML. Furthermore, 34+DR- cells from more than 80% of ECP CML patients were BCR/ABL mRNA- and Ph-negative and contained only BCR/ABL mRNA- and Ph-negative LTC-IC, whereas 34+DR- cells and LTC-IC from less than 40% of AP CML patients were BCR/ABL mRNA- and Ph-negative. In contrast to NL marrow, 34+DR+ cells from CML marrow, irrespective of clinical stage, contained large numbers of LTC-IC. CML 34+DR+ cells and LTC-IC were BCR/ABL mRNA- and Ph-positive. Since these studies suggested that a population of primitive progenitors that are Ph-negative can be selected from steady-state marrow in some ECP CML patients, we determined if similar results could be obtained when large quantities of marrow sufficient for transplantation are processed. We demonstrate that 1 to 3 x 10(5) BCR/ABL mRNA-negative 34+DR- cells/kg recipient body weight, containing only BCR/ABL mRNA-negative LTC-IC, can be obtained from a 2- to 2.5-L marrow collection by sequential COBE Spectra apheresis (COBE BCT, Lakewood, CO), CD34+ enrichment using the CEPRATE SC Cell-Concentrator (CellPro, Bothell, WA), and high-speed FACS. Thus, large-scale selection of a BCR/ABL mRNA- and Ph-negative 34+DR- cell population is possible in a fraction of chronic-phase CML patients, in whom these cells could be used to reconstitute the hematopoietic compartment following autologous transplantation.

Antigens, CD34↗

PubMed1996

Interferon-alpha restores normal beta 1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) progenitors show decreased adhesion to stroma and fibronectin (FN) through beta 1 integrin receptors. We have previously shown that interferon-alpha (IFN-alpha) restores beta 1 integrin-mediated adhesion of CML progenitors to stroma. Because beta1 integrins transmit proliferation inhibitory signals from the microenvironment to normal hematopoietic progenitors, we hypothesized that decreased integrin-mediated adhesion of CML progenitors contributes to their continuous proliferation when in contact with stroma and that IFN-alpha treatment, by restoring integrin-mediated adhesion, also restores integrin-mediated microenvironmental inhibition of CML progenitor proliferation. We show here that, in contrast to normal colony-forming cells (CFC), the percentage of malignant CML CFC in S-phase was not significantly reduced following coculture with stromal layers. However, IFN-alpha treatment resulted in a significant reduction in the proliferation of CML CFC on coculture with stroma. This effect was not because of a direct antiproliferative effect of IFN-alpha on CML CFC because the proliferation of IFN-alpha treated CML CFC kept in suspension culture was not reduced. We examined the role of restored signaling through beta 1 integrin receptors in IFN-alpha induced inhibition of CML progenitors in two sets of experiment. In the first set of experiments, we demonstrated that proliferation of IFN-alpha-treated CML CFC, but not untreated CML CFC, was significantly reduced following coculture with 33/66-kD and 75-kD FN fragments, recognized by alpha 4 beta 1 and alpha 5 beta 1 integrins respectively. In a second set of experiments, we demonstrate that direct stimulation of integrin receptors by crosslinking with blocking antibodies to alpha 4, alpha 5, and beta 1 integrins and secondary goat antimouse antibodies resulted in significant reduction in proliferation of normal and IFN-alpha treated CML progenitors but not untreated CML CFC. These studies indicate that CML hematopoietic progenitors are unresponsive to beta 1-integrin mediated proliferation inhibition and that IFN-alpha not only restores beta 1 integrin-mediated adhesion but also beta1-mediated microenvironmental inhibition of CML progenitor proliferation. These observations may explain, at least in part, the therapeutic efficacy of IFN-alpha in CML.

Bone Marrow↗

PubMed1996

Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.

Animals↗

PubMed1996

Activation-dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells.

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.

Antibodies, Monoclonal↗

PubMed1996

Autologous activated natural killer cells suppress primitive chronic myelogenous leukemia progenitors in long-term culture.

A graft-versus-leukemia effect has been well documented to prevent relapse in chronic myelogenous leukemia (CML) after allogeneic marrow transplantation. One type of lymphocytes that may contribute to this effect are natural killer cells (NK), which after activation with interleukin (IL)-2, exhibit a broad range of cytolytic activity against allogeneic and autologous cells. We have previously demonstrated that IL-2-activated NK (ANK) can be generated from blood of patients with CML and are benign in origin. Their proliferation and function, however, diminish with disease progression in CML, suggesting a role in tumor surveillance. We studied the effect of IL-2-activated NK (ANK) on normal and malignant primitive and committed progenitors in a novel long-term bone marrow culture (LTBMC) assay. Because ANK destroy marrow stromal layers, the use of classic stroma-dependent long-term cultures is not possible. Therefore, we used the stroma noncontact LTBMC system developed in our laboratory to analyze the effect of autologous ANK cells on primitive hematopoietic progenitors. Autologous ANK (CD56+/CD3-) were generated from the peripheral blood of 10 patients with chronic phase CML and from six normal individuals by culturing CD5/CD8-depleted mononuclear cells for 14 days in 1,000 U/mL IL-2. At the same time ANK cultures were initiated, sorted normal (CD34+/DR+) marrow populations were plated in Transwell inserts of the stroma noncontact culture. On day 15, hydrocortisone, which rapidly inhibits ANK function, was removed, and autologous ANK were added to the Transwell inserts with fresh LTBMC medium without hydrocortisone but supplemented with 1,000 U/mL IL-2. After 48 hours, the number of colony-forming cells (CFC) was enumerated in methylcellulose culture. To determine the effect of ANK on more primitive long-term culture-initiating cells (LTCIC), the IL-2-supplemented LTBMC medium was replaced with fresh hydrocortisone containing LTBMC medium, and cultures were maintained for an additional 5 weeks. We demonstrate that autologous ANK did not suppress normal CFC or LTCIC. In contrast, ANK from eight patients with CML with potent cytotoxicity against NK-sensitive (K562) NK-resistant (Raji) tumor targets exhibited an ANK dose-dependent suppression of both CFC and LTCIC. Interestingly, ANK from two patients with CML who exhibited diminished cytotoxicity also did not suppress autologous CFC and LTCIC. These studies indicate that ANK with potent major histocompatibility complex unrestricted cytotoxic activity suppress malignant hematopoiesis. This effect was not mediated by soluble factors and was absolutely dependent on direct cell-to-cell contact. We further demonstrate that the beta2 integrin receptor is involved in ANK recognition of CML targets. These observations support the use of autologous ANK therapy to prevent relapse of CML after autologous marrow transplantation or use of ANK to purge CML marrow for autologous transplantation.

Bone Marrow↗

PubMed1996

Direct contact with stroma inhibits proliferation of human long-term culture initiating cells.

Significantly less long-term culture initiating cells (LTC-IC) are recovered from cultures in which progenitors are cultured in contact with stroma (stroma-contact) than when cultured separated from stroma by a transwell (stroma-noncontact). This suggests that direct contact with stroma inhibits either proliferation or survival of LTC-IC. Using the membrane intercallating fluorochrome, PKH-26, we demonstrated that significantly less LTC-IC plated for 14 days in stroma-contact cultures proliferated than in stroma-noncontact cultures (16+/-7 vs 50+/-10%). Furthermore, when LTC-IC were sorted singly in stroma-contact cultures for 2 weeks, only 25+/-4% of individual LTC-IC progeny could initiate two secondary stromal cultures and had therefore proliferated, whereas 45+/-6% of single sorted LTC-IC progeny proliferated when cultured in stroma-conditioned medium without stromal feeder. However, LTC-IC survival was similar in both culture systems. Finally, proliferation inhibition occurred even when LTC-IC were cultured in contact with glutaraldehyde-fixed stroma, which is no longer capable of producing growth inhibitory or stimulatory cytokines. Thus, direct adhesive interactions between LTC-IC and stromal components inhibits their proliferation.

Bone Marrow Cells↗

PubMed1996

Refractory thrombocytopenia due to anti-PLA1 antibodies following autologous peripheral blood stem cell transplantation: case report and review of literature.

A 43-year-old woman developed life-threatening refractory thrombocytopenia following autologous peripheral blood stem cell transplantation for metastatic breast carcinoma. Anti-PLA1 antibodies were demonstrated in the patient's plasma. Treatment with intravenous immunoglobulin and transfusion of PLA1-negative blood products restored platelet responsiveness. PLA1 allo-antibodies should be considered as a possible cause of severe refractory thrombocytopenia in marrow transplant patients. It is important to make this diagnosis as the mainstay of treatment is the avoidance of blood products from PLA1-positive donors (98% of the population).

Adult↗

PubMed1995

A novel single-cell proliferation assay shows that long-term culture-initiating cell (LTC-IC) maintenance over time results from the extensive proliferation of a small fraction of LTC-IC.

We have previously shown that when adult marrow CD34+/HLA-DR- cells are cultured for 5 or 8 weeks in the presence of stroma-conditioned media with interleukin-3 (IL-3) and macrophage inflammatory protein-1 alpha (MIP-1 alpha), long-term culture-initiating cells (LTC-IC) are maintained but not expanded. However, if the same cultures are evaluated after 2 weeks, we show that LTC-IC expand 5.5- +/- 0.2-fold. Because expansion of LTC-IC is likely the result of a balance between proliferation and loss of LTC-IC, we hypothesized that, although LTC-IC proliferate in these cultures, loss of a fraction of LTC-IC underlies the lack of long-term expansion. To evaluate the fate of LTC-IC (proliferation, conservation, or loss), we performed PKH-26 labeling assays and developed a single LTC-IC proliferation assay. For PKH-26 labeling assays, CD34+/HLA-DR- cells were incubated with the membrane intercallating dye, PKH-26, before culture for 14 days in stroma-noncontact cultures + IL-3 + MIP-1 alpha. Progeny was reselected by fluorescence-activated cell sorting based on their PKH-26 fluorescence intensity. These studies showed that LTC-IC proliferate because 80% of LTC-IC at week 2 had 0.5 to 1 log lower fluorescence intensity than did freshly labeled CD34+/HLA-DR- cells. To further determine the fate of LTC-IC, we also developed a single LTC-IC proliferation assay. A population of CD34+/CD33- cells, highly enriched in LTC-IC, was sorted singly in stroma-conditioned media+IL-3 + MIP-1 alpha. After 5 weeks, the content of each well was divided equally over 8 secondary stroma-containing wells and cultured for 8 weeks to determine the capacity of the single-cell progeny to initiate 1 or more secondary stromal cultures. Progeny of single-sorted cells were able to initiate up to 8 secondary long-term cultures, demonstrating that LTC-IC proliferate in stroma-conditioned media+IL-3 + MIP-1 alpha. However, more than 65% of single-sorted LTC-IC were not conserved because their progeny could no longer initiate secondary long-term cultures. This finding indicates that, although stromal factors and IL-3 + MIP-1 alpha can induce proliferation of LTC-IC, failure to conserve a large fraction of LTC-IC results in lack of long-term expansion.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗

PubMed1995

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony-stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte-CSF.

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte-macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G-CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM-CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1995

Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages.

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC-IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth-inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.

Antigens, CD↗

PubMed1995

Autografting for chronic myelogenous leukemia.

Two thirds of patients with chronic myelogenous leukemia do not have suitable donors for allogeneic transplantation. As for other leukemias, autografting may potentially be curative, because normal Ph- hematopoietic stem cells persist in the marrow and blood of patients with chronic myelogenous leukemia. Several studies indicate that use of unpurged autologous blood or marrow grafts may extend survival for patients undergoing transplantation in chronic phase. Ex vivo or in vivo purging of chronic myelogenous leukemia marrow or blood prior to autografting may result in increased cytogenetic remissions after transplantation in those patients in whom the Ph+ clone can be eliminated. However, when the Ph+ clone cannot be eliminated, use of purged rather than unpurged autografts provides no advantage and may be associated with increased graft failure. Although sustained cytogenetic remissions have not been observed, autografting may result in a plateau in the survival curve not observed with conventional chemotherapy. Efforts are currently directed toward developing improved methods of purging as well as posttransplantation treatments directed against leukemic cells persisting after myeloablative therapy.

Bone Marrow Purging↗

PubMed1995

Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation.

In long-term bone marrow cultures, stroma-adherent progenitors proliferate significantly less than nonadherent progenitors. Thus, close progenitor-stroma interactions may serve to regulate or restrict rather than promote hematopoietic progenitor proliferation. We hypothesized that signaling through adhesion receptors on hematopoietic cells may contribute to the inhibition of proliferation observed when progenitors are in contact with stroma. We demonstrate that progenitors cultured physically separated from stroma in a transwell proliferate significantly more than progenitors adherent to stroma. Furthermore, proliferation of colony forming cells (CFC) is reduced after specific adhesion to stroma, metabolically inactivated glutaraldehyde-fixed stroma, stromal-extracellular matrix, or the COOH-terminal heparin-binding domain of fibronectin. Nonspecific adhesion to poly-L-lysine fails to inhibit CFC proliferation. That the VLA-4 integrin is one of the receptors that transfers proliferation inhibitory signals was shown using blocking anti-alpha 4 monomeric F(ab) fragments. Furthermore, when synthetic peptides representing specific cell attachment sites within the heparin-binding domain of fibronectin were added to Dexter-type marrow cultures, significantly increased recovery and proliferation of CFC was observed, suggesting that these peptides disrupt adhesion-mediated proliferation inhibitory events. Thus, negative regulation of hematopoiesis may not only depend on the action of growth inhibitory cytokines but also on growth inhibitory signals resulting from direct adhesive interactions between progenitors and marrow stroma.

Amino Acid Sequence↗

PubMed1995

Treatment of marrow stroma with interferon-alpha restores normal beta 1 integrin-dependent adhesion of chronic myelogenous leukemia hematopoietic progenitors. Role of MIP-1 alpha.

The mechanisms by which interferon-alpha (IFN-alpha) restores normal hematopoiesis in chronic myelogenous leukemia (CML) are not well understood. We have recently demonstrated that IFN-alpha acts directly on CML hematopoietic progenitors to restore their adhesion to marrow stroma by modulating beta 1 integrin receptor function. In the present study we examined the effect of IFN-alpha treatment of marrow stroma on subsequent adhesion of CML progenitors. Stromal layers were preincubated with IFN-alpha (10,000 microns/ml) for 48 h. Subsequent coincubation with CML progenitors for 2 h resulted in significantly increased adhesion of CML progenitors. We demonstrated that alpha 4 beta 1 and alpha 5 beta 1 integrin receptors were involved in the enhanced adhesion of CML progenitors, suggesting that IFN-alpha-treated stroma can upregulate CML integrin function. This effect is due, at least in part, to IFN-alpha-induced increased stromal production of the chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha), which upregulates beta 1 integrin-dependent adhesion of CML progenitors to stroma. Thus, IFN-alpha treatment of marrow stroma restores beta 1 integrin-dependent adhesion of CML progenitors, at least in part through induction of MIP-1 alpha production. These observations provide further insights into mechanisms by which IFN-alpha may restore normal hematopoiesis in CML.

Adipose Tissue↗

PubMed1994

Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor.

Close interaction of human hematopoietic progenitors with the bone marrow microenvironment is important for the ordered progression of human hematopoiesis. Progenitor cell adhesion to stroma has a complex molecular basis, involving various cell-extracellular matrix and cell-cell interactions. We have previously shown that adhesion of colony-forming cells (CFC) to fibronectin, present in stromal extracellular matrix, involves multiple sites, including two heparin-binding synthetic peptides (FN-C/H I and FN-C/H II) and the alpha 4 beta 1 integrin-binding peptide CS1. These synthetic peptides are located in close proximity in the type III repeat 14 and the immediately adjacent type IIIcs region of fibronectin. In the current study, we evaluate receptors expressed by CFC responsible for their adhesion to fibronectin. We show that the alpha 4 beta 1 integrin mediates adhesion to CFC to the peptides FN-C/H I and CS1. Adhesion of CFC to fibronectin is also mediated by proteoglycans, because removal of cell surface chondroitin-sulfate proteoglycans resulted in decreased adhesion of CFC to FN-C/ I and FN-C/H II. The core protein of this proteoglycan was identified by immunoprecipitation as a 90-kD member of the CD44 group of adhesion molecules. Interestingly, although the proteoglycan core protein failed to adhere to FN-C/H II affinity columns, anti-CD44 monoclonal antibodies blocked CFC adhesion to FN-C/H II, indicating that these monoclonal antibodies may interfere with core protein-mediated intracellular signalling. Finally, we show that CD44 and alpha 4 beta 1 may cooperate in establishing progenitor adhesion, because anti-CD44 antibodies potentiated the adhesion-inhibitory effects of suboptimal concentrations of anti-alpha 4 or anti-beta 1 monoclonal antibodies. These results provide a working model for progenitor cell recognition of fibronectin (and possibly the marrow micro-environment) in which the coordinated action of integrins and cell surface proteoglycans is necessary for cell adhesion. This model can now be used to study the complex relationship between progenitor cell adhesion and the regulation of their proliferation and differentiation.

Amino Acid Sequence↗

PubMed1994

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells.

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence-activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane ("stroma-noncontact" culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma-noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA-DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP-1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

Animals↗

PubMed1994

Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro.

Factors that induce proliferation of the human hematopoietic stem cell are ill-defined. Primitive hematopoietic progenitors can be maintained and differentiate in stroma-dependent, long-term bone marrow cultures (LTBMC), originally described by Dexter et al. (Dexter, T. M., L. H. Coutinho, E. Spooncer, C. M. Heyworth, C. P. Daniel, R. Schiro, J. Chang, and T. D. Allen. 1990. Molecular Control of Haemopoiesis). However, 70-80% of primitive progenitors capable of reinitiating secondary stromal cultures (LTBMC-initiating cells [IC]) are lost over a period of 5 wk in such cultures. We have recently described a novel "stroma-noncontact" culture system, in which hematopoietic progenitors are separated from the stromal layer by a 0.4-micron microporous filter membrane. Primitive progenitors in such cultures can not only differentiate into committed progenitors, but are also maintained to a greater extent than in "Dexter" cultures. However, still only 50% of the originally seeded LTBMC-IC are recovered at week 5. Since maintenance of primitive progenitors may depend not only on growth-promoting factors but also on factors that inhibit differentiation and/or proliferation, we evaluated the effect of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or "stem cell inhibitor" in combination with the growth-inducing factor interleukin 3 (IL-3) on the recovery of LTBMC-IC from stroma-noncontact cultures. We demonstrate that addition of MIP-1 alpha alone to stroma-noncontact cultures does not change the number of LTBMC-IC present after 8 wk, indicating that this factor may not directly inhibit or stimulate proliferation of primitive progenitors. Addition of the growth stimulatory cytokine, IL-3, alone results in exhaustion of LTBMC-IC after 8 wk of culture, possibly as a result of their terminal differentiation. However, LTBMC-IC can be maintained for at least 8 wk when grown in stroma-noncontact cultures supplemented with both MIP-1 alpha plus IL-3. This effect depends on soluble (ill-defined) stromal factors, and results from a direct interaction of these cytokines with the progenitor population or its progeny, but not the stroma.

Bone Marrow↗