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Traditionally, mesenchymal stem cells (MSCs) isolated from adult bone marrow were described as being capable of differentiating to various lineages including cartilage. Despite increasing interest in these MSCs, concerns regarding their safety, in vivo behavior and clinical effectiveness have restrained their clinical application. We hypothesized that MSCs have trophic effects that stimulate recycled chondrons (chondrocytes with their native pericellular matrix) to regenerate cartilage. Searching for a proof of principle, this phase I (first-in-man) clinical trial applied allogeneic MSCs mixed with either 10% or 20% recycled autologous cartilage-derived cells (chondrons) for treatment of cartilage defects in the knee in symptomatic cartilage defect patients. This unique first in man series demonstrated no treatment-related adverse events up to one year postoperatively. At 12 months, all patients showed statistically significant improvement in clinical outcome compared to baseline. Magnetic resonance imaging and second-look arthroscopies showed completely filled defects with regenerative cartilage tissue. Histological analysis on biopsies of the grafts indicated hyaline-like regeneration with a high concentration of proteoglycans and type II collagen. Short tandem repeat analysis showed the regenerative tissue only contained patient-own DNA. These findings support the novel insight that the use of allogeneic MSCs is safe and opens opportunities for other applications. Stem cell-induced paracrine mechanisms may play an important role in the chondrogenesis and successful tissue regeneration found. Stem Cells 2017;35:256-264., (© 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Using a combination of articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) has shown to be a viable option for a single-stage cell-based treatment of focal cartilage defects. However, there is still considerable debate whether MSCs differentiate or have a chondroinductive role through trophic factors. In addition, it remains unclear whether direct cell-cell contact is necessary for chondrogenesis. Therefore, the aim of this study was to investigate whether direct or indirect cell-cell contact between ACs and MSCs is essential for increased cartilage production in different cellular environments and elucidate the mechanisms behind these cellular interactions. Human ACs and MSCs were cultured in a 10:90 ratio in alginate beads, fibrin scaffolds, and pellets. Cells were mixed in direct cocultures, separated by a Transwell filter (indirect cocultures), or cultured with conditioned medium. Short tandem repeat analysis revealed that the percentages of ACs increased during culture, while those of MSCs decreased, with the biggest change in fibrin glue scaffolds. For alginate, where the lack of cell-cell contact could be confirmed by histological analysis, no difference was found in matrix production between direct and indirect cocultures. For fibrin scaffolds and pellet cultures, an increased glycosaminoglycan production and type II collagen deposition were found in direct cocultures compared with indirect cocultures and conditioned medium. Positive connexin 43 staining and transfer of cytosolic calcein indicated communication through gap junctions in direct cocultures. Taken together, these results suggest that MSCs stimulate cartilage formation when placed in close proximity to chondrocytes and that direct cell-cell contact and communication through gap junctions are essential in this chondroinductive interplay.

We performed a prospective phase II study to evaluate clinical safety and outcome in 48 patients with steroid-refractory grade II-IV acute graft-versus-host disease (aGVHD) treated with mesenchymal stromal cells (MSCs). Clinical outcomes were correlated to comprehensive analyses of soluble and cellular biomarkers. Complete resolution (CR) of aGVHD at day 28 (CR-28) occurred in 12 (25%) patients, CR lasting >1 month (CR-B) occurred in 24 (50%) patients. One-year overall survival was significantly improved in CR-28 (75 versus 33%, P=0.020) and CR-B (79 versus 8%, P<0.001) versus non-CR patients. A six soluble biomarker-panel was predictive for mortality (HR 2.924; CI 1.485-5.758) when measured before MSC-administration. Suppression of tumorigenicity 2 (ST2) was only predictive for mortality 2 weeks after but not before MSC-administration (HR 2.389; CI 1.144-4.989). In addition, an increase in immature myeloid dendritic cells associated with decreased mortality (HR 0.554, CI 0.389-0.790). Patients had persisting T-cell responses against defined virus- and leukemia-associated antigens. In conclusion, our data emphasize the need to carefully assess biomarkers in cohorts with homogeneous GVHD treatments. Biomarkers might become an additional valuable component of composite end points for the rapid and efficient testing of novel compounds to decrease lifecycle of clinical testing and improve the success rate of phase II/III trials.

The evolution of articular cartilage repair procedures has resulted in a variety of cell-based therapies that use both autologous and allogeneic mesenchymal stromal cells (MSCs). As these cells are increasingly available and show promising results both in vitro and in vivo, cell-based strategies, which aim to improve ease of use and cost-effectiveness, are progressively explored. The use of MSCs in cartilage repair makes it possible to develop single-stage cell-based therapies. However, true single-stage procedures rely on one intervention, which will limit cell sources to fraction concentrates containing autologous MSCs or culture-expanded allogeneic MSCs. So far, it seems both autologous and allogeneic cells can safely be applied, but clinical studies are still ongoing and little information on clinical outcome is available. Further development of cell-based therapies may lead to clinical-grade, standardized, off-the-shelf products with easy handling for orthopedic surgeons. Although as of yet no preclinical or clinical studies are ongoing which explore the use of induced pluripotent stem cells for cartilage repair, a good manufacturing practice-grade induced pluripotent stem cell line might become the basis for such a product in the future, providing that cell fate can be controlled. The use of stem cells in clinical trials brings along new ethical issues, such as proper controls and selecting primary outcome measures. More clinical trials are needed to estimate detailed risk-benefit ratios and trials must be carefully designed to minimize risks and burdens for patients while choosing outcome measures that allow for adequate comparison with results from similar trials. In this review, we discuss the different aspects of new stem cell-based treatments, including safety and ethical issues, as well as provide an overview of current clinical trials exploring these approaches and future perspectives.

Background: Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow-derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of bone marrow mononuclear cells (BMMNCs) in patients with severe, nonrevascularizable limb ischemia can prevent major amputation., Methods and Results: The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, nonrevascularizable limb ischemia. Patients were randomly assigned to repetitive (3 times; 3-week interval) intra-arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, ie, major amputation at 6 months, with major amputation rates of 19% in the BMMNC versus 13% in the placebo group (relative risk, 1.46; 95% confidence interval, 0.62-3.42). The safety outcome (all-cause mortality, occurrence of malignancy, or hospitalization due to infection) was not significantly different between the groups (relative risk, 1.46; 95% confidence interval, 0.63-3.38), neither was all-cause mortality at 6 months with 5% versus 6% (relative risk, 0.78; 95% confidence interval, 0.22-2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups., Conclusions: Repetitive intra-arterial infusion of autologous BMMNCs into the common femoral artery did not reduce major amputation rates in patients with severe, nonrevascularizable limb ischemia in comparison with placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC and the placebo group, as well, underlines the essential role for placebo-controlled design of future trials., Clinical Trial Registration Url: http://www.clinicaltrials.gov. Unique identifier: NCT00371371., (© 2015 American Heart Association, Inc.)

In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.

SUMMARY: In this review, the appropriate legislation on the expansion of multipotent mesenchymal stromal cells (MSCs) in Europe is described. The collection of cells and the manufacturing of MSCs are being regulated by European Directives (EUDs). Recently, the Regulation on Advanced Therapies Medicinal Products (ATMPs) is being published, which is of importance for the production of MSCs in Europe, and this legislation is not yet ready, but it is in its final stage. MSCs are currently being used in clinical trials, mostly in academic hospitals, for patients suffering from a wide variety of diseases. Companies (small and medium-sized enterprises) are becoming more and more involved in the production of MSCs for human use, and since marketing authorisation is the scope of the Regulation it was decided to install a Committee on Advanced Therapies (CAT) within European Medicines Agency (EMEA). This CAT will formulate a draft opinion on quality, safety and efficacy of ATMPs and will have an advisory and scientific role for the Committee for Medicinal Products for human use. The aim of this review is to outline the current legislation which is important for the manufacturing of MSCs.

Background: Microvascular endothelial cells (MVEC) derived from s.c. fat are seeded on vascular grafts to prevent early occlusion. We have demonstrated the presence of contaminating cells contributing to MVEC seeding-related intimal hyperplasia in MVEC isolates from fat tissue. We found that cell isolates additionally purified after the isolation process, were associated with a reduced thrombogenicity and development of intimal hyperplasia in vitro. A combination of 11Fibrau (F11)- and CD14-coated Dynabeads was used to deplete the contaminating cells, fibroblasts, and monocytes/macrophages. Unfortunately, clinical-grade F11 is not available, and thus cannot be used for clinical practice. CD34 selection with clinical-grade products is widely used for the isolation of hematopoietic progenitors, and endothelial cells (EC) express CD34 on their surfaces. The aims of this study were to test the effectiveness of two different CD34-selection techniques for purification of MVEC, and to compare the results with those of the F11/CD14-method., Methods: Liposuction fat was enzymatically digested and centrifuged twice to remove adipocytes and collagenase. CD34 selection was performed using the commercially available methods from Nexell or Miltenyi. Both techniques were modified for our use. The purity after isolation and culture, and recovery were determined by flow-cytometry (CD31-expression) and compared with that of cells purified with the F11/CD14-method., Results: Besides MVEC, the contaminating fibroblasts and macrophages/monocytes weakly expressed the CD34 Ag. Enrichment of MVEC was not successful with the Miltenyi method. Variations in neither the dose of Ab nor the use of direct selection and different separation programs improved the results. With the Nexell method, MVEC were enriched to 86%, a comparable purity to that obtained with the F11/CD14-method. However, a lower recovery was achieved with the Nexell method., Conclusion: Enrichment of MVEC could be achieved with a modified protocol of the clinical grade CD34(+) selection method from Nexell, but not with the CD34 method from Miltenyi.

Here, the influence of T vs T and B cell depletion on the incidence of EBV-associated lymphoproliferative disorder (EBV-LPD) after bone marrow transplantation (BMT) from a matched unrelated donor (MUD) is analyzed. From 1982 to 1997 the soy bean agglutinin/sheep red blood cell (SBA/SRBC) method was used for T cell depletion. This technique is well established, but the use of SRBC has a risk of transmitting prions or viruses. Therefore, a new T cell depletion method was introduced, using CD2 and CD3 monoclonal antibodies (CD2/3 method) instead of SRBC. Unfortunately, this led to an unexpected high number of EBV-LPDs in patients receiving transplants from MUDs. SBA depletion was reintroduced and combined with the CD2/3 method (SBA/CD2/3) in this patient population, later replaced by B cell-specific (CD19 and CD22) antibodies (CD3/19/22 method). The number of T (x 10(5)/kg) and B (x 10(5)/kg) cells in the graft was 1.5 +/- 0.8 and 2 +/- 1 (T/B ratio 0.75), 2.2 +/- 2.0 and 41 +/- 21 (ratio 0.055), 5.0 +/- 0.0 and 2 +/- 1 (ratio 2.5), 2.5 +/- 1.2 and 10 +/- 6 (ratio 0.25) using the SBA/SRBC, CD2/3, SBA/CD2/3 and CD3/19/22 techniques, respectively. When B cell depletion was performed (SBA/SRBC, SBA/CD2/3, CD3/19/22) four out of 31 patients (13%) receiving a BMT from a MUD developed an EBV-LPD. Without B cell depletion (CD2/3) this occurred in five out of seven patients (71%) (P < 0.05). A T/B cell ratio in the graft of > or = 0.25 seems sufficient to significantly reduce the incidence of EBV-LPD after BMT from MUDs.

To analyse the involvement of a possible numerical or qualitative stem cell defect in the development of sustained graft failure after autologous transplantation, we have determined the graft content of CD34+ nucleated cells, colony-forming cells and cobblestone area-forming cell subsets, as well as transplant ability to produce progenitors using the long-term culture colony-forming cell (LTC-CFC) assay. We evaluated material from the graft reference ampoules of 13 graft failure patients after bone marrow transplantation (BMT), four graft failure patients and four isolated thrombocytopenia patients after peripheral blood stem cell transplantation (PBSCT). We compared these data with those from six successfully engrafted BMT patients and 20 engrafted PBSCT patients respectively. In the BMT setting, the LTC-CFC 6-week assay represented a highly significant graft failure predictor. In the PBSCT setting, the total number of 2-week and 6-week LTC-CFCs transplanted per kg bodyweight (BW) showed the highest significant difference between the engrafted and the graft failure patients, as well as between the engrafted patients and the patients suffering from isolated thrombocytopenia after transplantation. These data show that the ability of a graft to generate progenitors in vitro rather than the number of primitive progenitors transplanted can have prognostic value for post-transplant haematological reconstitution.

Objective: In our laboratory, we have developed an immunorosette technique for the depletion of T cells from bone marrow transplants. Tetrameric complexes of monoclonal antibodies are able to form very stable immunorosettes, which are efficiently depleted with the aid of a blood cell separator. Major improvements over the original sheep red blood cell depletion are the use of human (patient or donor derived) erythrocytes instead of sheep-derived cells, and the possibility of using a closed system for separation in a cell separator. In contrast to bone marrow, mobilized haematopoietic stem cell transplants obtained after leucocytapheresis contain higher numbers of T cells. Therefore, a different approach is necessary., Method: We have used two CD34 selection systems (Isolex 300SA and the Clinimacs) to perform T-cell depletions from peripheral blood stem cell (PBSC) transplants., Results: Immunorosette T-cell depletion, with CD2/CD3 tetrameric complexes, of bone marrow transplants resulted in a mean 2.5 log depletion of T cells with a yield of 50% of the CD34+ cell population. Stem cell selection of PBSC transplants using one of the CD34 selection procedures resulted in a 4.5 log depiction of T cells for both systems, but with different results for the recovery of CD34+ cells. An increased yield of CD34+ cells was obtained with the Clinimacs procedure (57.9+/-9.0%) in comparison to the Isolex procedure (40.1+/-12.5%)., Conclusion: Our own immunorosette depletion technique and the two tested CD34 selection methods for stem cell transplants both resulted in a very efficient T-cell depletion with the recovery of 40-60% of the CD34 haematopoietic stem cells present in the transplant.

We have designed and used an in vitro bone marrow cell culturing system for investigating pharmacodynamic interactions between platinum anti-cancer drugs and taxanes. With this system, in which the bone marrow progenitor cell CFU-GM is proliferating and differentiating into granulocytes and monocytes, we could show a strong antagonistic cytotoxicity of the combination carboplatin and Taxotere, in three different schedules, and of the combination cisplatin and Taxol, in two out of the three schedules tested. Modulation of intracellular platinum drug accumulation in granulocytes and monocytes does not seem to be a plausible explanation for the observed antagonism. In vitro co-incubation of granulocytes/monocytes with the combination carboplatin and Taxotere did not reveal an effect of Taxotere on intracellular platinum accumulation. Although Taxol reduced intracellular cisplatin levels by 12%, this effect was not significantly different from the co-incubation of cisplatin with Cremophor EL, the solvent for paclitaxel in Taxol. The toxicity data obtained in this study seem to be in accordance with recent clinical trials where combination therapies with platinum drugs and taxanes resulted in marked reductions in myelosuppression in patients. Therefore, these types of assays could be useful as to the assessment of bone marrow toxicities of clinically important drug combinations.

The aim of this study was to develop an inexpensive method for reducing the number of differentiated cells from granulocyte colony-stimulating factor-mobilized leukocytapheresis products (LPs) containing peripheral blood stem cells. Analysis of LPs showed the presence of significant numbers of monocytes and myeloid cells. The myeloid cells represented largely immature stages of the granulocyte lineage (myelocytes and metamyelocytes). We investigated whether these cells could be selectively depleted by filtration over nylon wool. Filtration of LP samples over nylon wool in a medium containing fetal calf serum resulted in variable but on average low yields of CD34+ cells (48 +/- 30%; n=13) and strongly variable depletions of myeloid cells. The adherence of CD34+ cells to the polyamide fiber was partially mediated by activated platelets that were present in the LPs. Removal of platelets by counterflow centrifugal elutriation before filtration resulted in increased yields of CD34+ cells in the filtrates (65 +/- 13%; n=10). The yield of progenitor cells was similarly enhanced when trisodium citrate, a chelating substance, was added to the filtration medium. Adherence of the myeloid cells to the nylon fiber was promoted by preincubation of the columns with human immunoglobulin (Ig) (2 mg/mL). Small-scale filtrations of LP samples in the presence of trisodium citrate over columns with Ig-coated nylon wool resulted in removal of 96 +/- 4% of the monocytes and 74 +/- 18% of the myeloid cells, with a yield of 71 +/- 15% CD34+ cells and 67 +/- 10% granulocyte-monocyte colony-forming units (CFU-GM) (n=23). There was no loss of primitive stem cells during the procedure: the yield of late-appearing cobblestone area-forming cells (CAFCs, week 6) was 110 +/- 30% (n=4). CFU-GM production per CAFC-derived clone was unchanged upon filtration, indicating that the quality of stem cells was not affected. Moreover, the proportions of CD34+ cells expressing a primitive immunophenotype (CD38low or Thy-1+) were unchanged after filtration. Further enrichment of progenitor cells was obtained by separation of LP samples by elutriation before filtration. The combination of these two techniques resulted in complete removal of platelets, 89 +/- 7% depletion of erythrocytes, and 91 +/- 6% reduction of leukocytes, with a 50% yield of CD34+ cells (n=14). In conclusion, we have developed a rapid filtration technique by which monocytes and myeloid cells can be depleted from LP samples, with only minor loss of colony-forming cells and complete recovery of primitive stem cells.

In our laboratory, a two-step procedure is used for purging precursor B ALL from autologous bone marrow grafts of children in second bone marrow remission. An immunorosette depletion method with CD19 and CD22 MAbs is followed by one cycle of complement-mediated cell lysis with CD9 and CD10 MAbs. The aim of the present study was to determine if the efficacy of this procedure could be further enhanced by including CD20 and CD72 MAbs in the current protocol. Leukemia-contaminated remission bone marrow was simulated by mixing cell line cells and normal bone marrow cells. The efficacy of purging of malignant cells was determined by culturing the cells in a limiting dilution assay. The effect of including CD20 and CD72 in the immunorosette depletion was limited. In contrast, when these MAbs were added during complement-mediated cell lysis, a significant increase in depletion of tumor cells was observed. This was true when complement lysis was carried out alone (0.4 versus 3.0 log depletion for Ros cells) and when it was preceded by immunorosette depletion (2.7 versus 4.1 log depletion for Ros cells). The loss of hematopoietic progenitor cells was not greater than with the current purging protocol.

The suitability of counterflow centrifugal elutriation (CCE) for reduction of the number of non-stem cells in autologous G-CSF-mobilized peripheral blood stem cell (PBSC) transplants was investigated. By cell size-monitored CCE, small cells could be rapidly separated from the haemopoietic progenitor cells present in leukapheresis product (LP) samples. The large cell fraction contained an average 86 +/- 25% of the CD34+ cells and 76 +/- 20% of the granulocyte-macrophage progenitors (CFU-GM) loaded into the separation chamber, and was depleted of 75 +/- 18% of the lymphocytes, 89 +/- 7% of the erythrocytes and 98 +/- 2% of the platelets (n = 21). Due to the presence of high numbers of large immature myeloid cells, which co-elutriated with progenitor cells, enrichment of CD34+ cells in the large cell fraction was only modest (average 1.8 times). No indication of preferential co-elutriation of primitive stem cells with the small cells was obtained. There was no difference in expression of CD38 or Thy-1 on CD34+ cells between the two elutriation fractions. Frequencies of cobblestone-area-forming cells (CAFC) week 6, which are considered to represent cells with long-term repopulating ability, were reduced in the small cell fractions as compared to those in the unseparated samples and the large cell fractions. On average, 100% of CAFC week 6 were recovered in the large cell fractions (n = 5). In conclusion erythrocytes, platelets and 40-50% of leucocytes can be depleted from G-CSF-mobilized PBSC samples by CCE with an almost complete recovery of both clonogenic and primitive stem cells.

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR-negativity) or not. In contrast, none of the five patients with PCR-negative grafts before purging relapsed (P = 0.0008). One patient died due to transplant-related toxicity. Of the remaining 23 patients, nine patients received a PCR-positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR-negative after purging (P = 0.0072). Two of eight PCR-positive BM grafts could be purged to PCR-negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.

Improvement of the collection efficiency (CE) of the Fenwal CS3000 plus in collecting circulating progenitor cells (CPC) might diminish the number of leukapheresis procedures (LP) required to obtain the CPC required to assure engraftment. We analyzed whether adjustment of the optical setting (location 71,L71) to the number of MNC present in the peripheral blood could enhance the CE of the MNC. Thirty-five patients underwent 121 LP with an adjusted L71. We compared the results retrospectively with 26 LP performed with a fixed L71 (1:100) in 12 patients. The CPC were mobilized with chemotherapy followed by subcutaneous administration of granulocyte colony-stimulating factor (G-CSF) in both groups. Adjustment of the L71 did neither improve the CE of the MNC, the estimated CE of CD34+ cells nor diminished granulocyte contamination. For the total 121 LP with an adjusted L71 and for the total 26 LP with a fixed L71 the mean CE of MNC were, respectively, 44.6 +/- 18.3% and 46.4 +/- 14%. The mean granulocyte contamination, determined by manual white blood cell differentiation, was 1.7 +/- 2.3% for the adjusted L71 group and 2.3 +/- 3 for the fixed L71 group. There was no difference in the median number of LP required to obtain 3 x 10(6) CD34+ cells/kg between both groups. We found a weak significant correlation between WBC and pre-LP MNC count and the CE of MNC (r = 0.36, P = 0.012, resp.r = 0.33, P = 0.023), but no correlation between the CE of MNC and the estimated CE of CD34+ cells (r = 0.24, P = 0.113). In conclusion, adjustment of the L71 to the MNC did not improve the CE of MNC of the Fenwal CS3000. The lack of correlation between the CE and MNC and the estimated CE of CD34+ cells should be further explored.

The mobilisation characteristics of ifosfamide and etoposide followed by Granulocyte Colony-Stimulating Factor fGCSF, filgrastim) were analysed in 17 patients with malignant lymphoma and 24 patients with solid tumours, with respect to the optimum time to harvest progenitor cells and to the yields of progenitor cells that could be achieved. In addition, we analysed patient characteristics which could influence the size of the progenitor cell harvest. Clinical parameters which were co-related with the size of the circulating progenitor cells (CPC) harvests were: the dose of G-CSF, dose of if osfamide, sex, age, diagnosis and extent of pretreatment. CPC were mobilised with 3 g/m2 (n = 11) or 4 g/m2 (n = 30) ifosfamide on day 1 and etoposide 100 mg/m2/day, on days 1-3 i.v., followed by daily s.c. injections with filgrastim 5 micrograms/kg (n = 26) or 10 micrograms/kg (n = 15) from day 4. The maximal progenitor cell harvest was achieved on either day 12 or day 13 after the start of the ifosfamide/etoposide course. The median number of leukaphereses necessary to harvest the target quantity of 3 x 10(6) CD34+ cells/kg body weight was 1 (range 1-9). Thirteen/41 (32%) of the patients did not achieve the target yield of 3 x 10(6) CD34+ cells/kg. By multivariate analysis, the dose of GCSF and prior irradiation were associated with the number of progenitor cells harvested, while all other parameters, induding the dose of if osfamide and number of previous chemotherapy courses, were not. Sixteen patients received two or more mobilisation courses. Despite the fact that the same mobilisation schedule was used, the progenitor cell yields after the first mobilisation course did not predict the results after the subsequent mobilisation courses, indicating that unknown transient factors may significantly influence the CPC yield.

In this study we investigated the applicability of 99mTc-labeled CD19 monoclonal antibody (mAb) for tumor imaging in patients with B cell non-Hodgkin's lymphoma. A 1-mg sample of murine CD19 mAb was labeled with approximately 550 MBq [99mTc]pertechnetate. The labeled mAb was administered i.v. to seven patients, four without and three with pretreatment with 10 mg unlabeled CD19 mAb. The number of circulating B cells was decreased by 44 +/- 5% 1 h after injection of the radiolabeled mAb. Peripheral B cells were coated with CD19, resulting in partial modulation of CD19, most pronounced in the three pretreated patients. Whole-body images were obtained with a gamma camera and compared with results obtained by conventional imaging techniques. Initially, blood-pool activity dominated, whereas 24 h after injection the radioactivity was mainly located in the spleen, kidneys and liver. In two patients, a lesion in the spleen appeared as an unlabeled spot. In one patient, a lesion in the femur, which was detected by computed tomography (CT) and gallium-67 scans, was also seen on the CD19 scan from 1 h after administration of the radioimmunoconjugate onwards. Good imaging of bone marrow infiltration was observed in one of three patients. Lymph node involvement was not observed in any of the patients in whom affected lymph nodes were detected by CT or gallium-67 scan. In conclusion, in the present study radioimmunodetection with 99mTc-labeled CD19 mAb was found to be inferior to CT and gallium-67 scanning in the diagnosis of patients with B cell non-Hodgkin's lymphoma.

Purpose: To determine the feasibility and safety of multiple, closely timed courses of high-dose cyclophosphamide, thiotepa, and carboplatin (CTC) with peripheral-blood progenitor-cell transplantation (PBPCT)., Patients and Methods: Forty-eight patients with advanced cancer were scheduled to undergo either two or three courses of CTC with PBPCT. All PBPCs were harvested before high-dose therapy began. Full-dose CTC courses incorporated cyclophosphamide (6,000 mg/m2), thiotepa (480 mg/m2), and carboplatin (1,600 mg/m2) divided over days -6, -5, -4, and -3. Tiny CTC courses (tCTC) contained 67% of the doses of each of these agents. Second or third courses of CTC or tCTC began on day 28., Results: A sufficient number of PBPC could be harvested from all but two patients. Thirty-five first full-dose courses of CTC were given, 28 second courses, and 10 third courses. Second courses could be given on time and at full dose in 80% of the patients, but there was one toxic death from venoocclusive disease (VOD). Only four of 12 patients scheduled to receive three courses of full-dose CTC could be treated at the time and dose planned. There were three toxic deaths: one of VOD, one of sepsis, and one of hemolytic uremic syndrome (HUS). Eight patients were scheduled to receive three courses of tCTC. Eight first, seven second, and six third courses were given. One of the third courses had to be delayed and one had to be reduced in dose., Conclusion: A sufficient number of PBPCs for two or three transplantations can be harvested from most patients without much difficulty before high-dose therapy. Two full-dose CTC courses or three tCTC courses can be given safely and with acceptable toxicity at 5-week intervals. Organ toxicity rather than bone marrow toxicity has become dose-limiting for alkylating agents.

In this study we describe a fast and sensitive method using three-colour immunofluorescence for the detection of cells with phenotypes that are rare in normal bone marrow (BM) but occur frequently in children with precursor B acute lymphoblastic leukaemia. We show that, in the first year after initiation of therapy, in 17/18 patients (10 patients were analysed after first diagnosis and nine patients after first BM relapse; one patient was analysed on both occasions) the percentage of CD10+ CD19+ cells and CD20- CD22+ cells in the CD34+ cell population indicated the likelihood of relapse. A suppression of cells expressing these phenotypes after initiation of therapy was followed by an outgrowth of normal precursor B cells after 12 months. Therefore this early test for impending relapse (which occurred 10-28 months after starting chemotherapy) was only applicable in the first year after beginning the treatment. However, despite this predictive value, comparison of fluorescence data with PCR results obtained from the same BM sample indicated that only a subpopulation of the CD34+ CD10+ CD19+ and CD34+ CD20- CD22+ cells above the determined threshold value represented malignant cells. A large prospective study to confirm the predictive value of this three-colour immunofluorescence assay is warranted.

Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37 degrees C, 20%-25% of initially cell-bound (125)I-CD19 mAb and (125)I-CD22 mAb was degraded in B cells, whereas almost no degredation occurred after binding of (125)I-CD20 mAb. For B cells expressing Fcgamma receptor II (FcgammaRII), isotype-dependent degradation was noted as the CD19 IgGl mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with (125)I-CD19 mAb or of CD20 with (125)I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcgammaRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant b cells and consequently result in an enhanced therapeutic effect in vivo.

Purpose: To study whether there is a relationship between transplanted cell dose and rate of hematopoietic recovery after peripheral-blood stem-cell (PBSC) transplantation, and to obtain an indication whether specific subsets of CD34+ cell populations contribute to rapid recovery of neutrophils or platelets., Patients and Methods: Based on data from 59 patients, we calculated for each day after PBSC transplantation the dose of CD34+ cells that resulted in rapid recovery of either neutrophils or platelets in the majority (> 70%) of patients. Using dual-color flow cytometry, subsets of peripheral-blood CD34+ cells were quantified and the numbers of CD34+ cells belonging to each of the reinfused subsets correlated with hematopoietic recovery following high-dose chemotherapy., Results: The calculated threshold values with a high probability of engraftment showed a steep dose-effect relationship between CD34+ cell dose and time to recovery of both neutrophils or platelets. Predominantly CD34+ cells with the phenotype of myeloid precursors were mobilized. A minority of CD34+ cells expressed the erythroid and megakaryocytic lineage-associated antigens and a low but distinct population of CD34+ cells expressed antigens associated with multipotent stem cells. Analysis showed that the number of CD34+CD33- cells (r = -.74, P < .05), as well as the number of CD34+CD41+ cells (r = -.81, P < .005), correlated significantly better with time to neutrophil and platelet recovery, respectively, than with the total number of CD34+ cells (r = -.55 and r = -.56, respectively)., Conclusion: The numbers of CD34+CD33- cells and CD34+CD41+ cells may help to predict short-term repopulation capacity of PBSCs, especially when relatively low numbers of CD34+ cells per kilogram are reinfused.

In 29 chemotherapy-naive patients with stage II-III breast cancer, peripheral blood stem cells (PBSCs) were mobilised following fluorouracil 500 mg m-2, epirubicin 90-120 mg m-2 and cyclophosphamide 500 mg m-2 (FEC) and granulocyte colony-stimulating factor (G-CSF; Filgrastim) 300 microgram s.c. daily. In all but one patient, mobilisation was successful, requiring three or fewer leucocytopheresis sessions in 26 patients; 28 patients subsequently underwent high-dose chemotherapy consisting of carboplatin 1600 mg m-2, thiotepa 480 mg m-2 and cyclophosphamide 6 g m-2 (CTC) followed by PBSC transplantation. Haemopoietic engraftment was rapid with a median time to neutrophils of 500 x 10(6) l(-1) of 9 days (range 8-10) in patients who received G-CSF after PBSC-transplantation; platelet transfusion independence was reached within a median of 10 days (range 7-16). Neutropenic fever occurred in 96% of patients. Gastrointestinal toxicity was substantial but reversible. Renal, neural or ototoxicity was not observed. Complications related to the central venous catheter were encountered in 64% of patients, with major vein thrombosis occurring in 18%. High-dose CTC-chemotherapy with PBSC-transplantation, harvested after mobilisation with FEC and G-CSF, is reasonably well tolerated without life-threatening toxicity and is a suitable high-dose strategy for the adjuvant treatment of breast cancer.

Background: Peripheral blood progenitor cell transplantation is rapidly replacing autologous bone marrow transplantation as hematological support after high-dose chemotherapy for lymphoma or solid tumors. Controversy exists concerning the number of progenitor cells required for rapid and sustained bone marrow recovery, and as to which of the widely available methods for estimating this number should be employed., Methods: Forty consecutive patients with solid tumors or lymphomas received high-dose chemotherapy followed by autologous peripheral stem cell reinfusion. All stem cell harvests had been performed after mobilization with standard-dose chemotherapy followed by 300 micrograms G-CSF daily. Hematopoietic reconstitution was studied in relation to pertinent patient characteristics, to the size of the graft (in terms of the total number of mononuclear cells (MNC), the number of granulocyte/macrophage colony-forming units (CFU-GM) and the number of CD34+ cells, and to the use of G-CSF after stem cell reinfusion., Results: Both the numbers of CFU-GM and CD34+ cells reinfused, but not those of the MNC, correlated with granulocyte and platelet recovery. Patients who received at least 5 x 10(6) CD34+ cells/kg body weight achieved platelet transfusion independence on day 12 after reinfusion (range: day 7-37), significantly earlier than patients who had received less (p = 0.001). Thirty patients who received G-CSF (300 micrograms s.c. daily) after reinfusion achieved granulocyte recovery (> or = 500 x 10(6)/l) on day 9 (range: day 8-12), while this took a median of 15 days (range: day 10-28) in 10 consecutive patients not receiving G-CSF (p = 0.0003). In one patient who had received 1.4 x 10(6) CD34+ cells/kg, secondary bone marrow failure developed 3 months after transplantation. Reinfusion of cryopreserved autologous bone marrow was followed by prompt recovery., Conclusion: Peripheral stem cells, mobilized by moderate-dose chemotherapy and G-CSF, lead to rapid and durable engraftment after high-dose chemotherapy when at least 3-5 x 10(6) CD34+ cells/kg are reinfused. Lower numbers may also be satisfactory, but are associated with slower granulocyte and platelet recoveries. A moderate dose of G-CSF after reinfusion significantly hastens granulocyte recovery without interfering with platelet recovery.

Background: Studies on radiolabeled monoclonal antibodies (MoAb) have dealt mainly with single antibodies. However, major differences may exist among different radiolabeled MoAb that bind to the same antigen and between switch variants of the same antibody. This study evaluates and compares a series of radiolabeled MoAb of different specificities, subclasses, and isotypes applicable in treatment of patients with B cell malignancies., Methods: MoAb were iodinated with iodogen. Immunoreactivity was determined in cell binding assays. Scatchard analyses were performed to determine association constants of radiolabeled MoAb and to measure antigen density on malignant B cells in various differentiation stages. The fate of the MoAb after antigen binding in vitro was studied by modulation and internalization experiments., Results: All MoAb tested could be iodinated efficiently and displayed association constants of 0.9 x 10(9)M-1 or higher. Immunoreactivity of radiolabeled MoAb ranged from 62-79%, except for the immunoglobulin (Ig)-M MoAb CLB-MD20.2, which had an immunoreactivity of 43%. The highest number of binding sites was detected for the CD20 MoAb (12 x 10(3) - 355 x 10(3), whereas the expression of antigens recognized by the CD22 MoAb was lowest on all cell types tested (4 x 10(3) - 26 x 10(3)). The MoAb CD19 and CD22 both induced modulation, whereas the CD20 MoAb did not. Modulation induced by the CD19 MoAb was caused by internalization. The rate of internalization was isotype-dependent and, for CD19-IgG1, partly mediated by Fc gamma ReceptorII., Conclusions: Radiolabeled B cell MoAb tested in this study are promising for use in radioimmunotherapy. For therapy with the radioisotope iodine-131, the IgG2a and IgG2b CD19 MoAb are more suitable than CD19-IgG1, because of their slower modulation and internalization rate.

In this study, we investigated the effect of recombinant human interleukin-6 (IL-6) on colony-forming cells for granulocytes and macrophages (CFU-GM) cultured in suspension. IL-6 when used alone did not induce proliferation of highly purified CD34+ human hematopoietic progenitors. Moreover, no influence of IL-6 was observed on the proliferation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte (G)-CSF. However, a marked survival enhancement (GM-CSF 228 +/- 42%, p < 0.01, and G-CSF 137 +/- 9%, p < 0.05) was observed when CD34+ cells were preincubated with IL-6 for 6 days. This survival effect became even more pronounced under serum-poor conditions (GM-CSF 380 +/- 80%, p < 0.01, and G-CSF 180 +/- 20%, p < 0.01) and could also be demonstrated at the single cell level in a colony-forming assay. By analysis of subpopulations of CD34+ bone marrow (BM) cells selected on the basis of CD45RO expression, the observed IL-6-mediated survival effect was found to be restricted to the CFU-GM containing CD45RO- subset. Our data show that IL-6 is a survival factor for CFU-GM.

In this study we have made a detailed analysis of growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF], and macrophage colony-stimulating factor [M-CSF])-induced proliferation and differentiation of highly purified CD34+ committed human myeloid progenitor cells in suspension cultures. The results were compared with colony formation in semisolid medium. Proliferation in suspension cultures was determined by means of incorporation of [3H]thymidine, differentiation by flow cytometric immunophenotyping using a panel of monoclonal antibodies against monomyeloid antigens, and by morphology. A good correlation was found between the number of granulocyte-macrophage colony-forming units (CFU-GM) in semisolid medium and [3H]thymidine incorporation in suspension (r = 0.82), both assessed at day 11. Moreover, the frequency of proliferating cells as determined in suspension cultures by limiting dilution analysis was similar to the frequencies of CFU-GM as measured in semisolid medium. Studies on GM-CSF- and G-CSF-induced cell-growth kinetics revealed distinct proliferation patterns. Immunophenotypically the subsequent induction of the mature granulocytic antigens CD15 and CD67 was observed to be accompanied by a gradual loss of the HLA-DR antigen, whereas little monocytic differentiation was observed. M-CSF, although inducing no colony formation of CD34+ cells and minimal proliferation in suspension, induced monocytic differentiation, demonstrated by the expression of HLA-DR, CD14, and CD36 in the absence of CD15 and CD67. The observed immunophenotypical profiles were confirmed by the results of cytological characterization. Thus, the combined measurement of growth factor-induced proliferation and differentiation of progenitor cells in suspension cultures can be a useful alternative for the CFU-GM assay. Moreover, because small numbers of cells are required, it allows for detailed studies on cell-growth kinetics and developmental stages within the granulocytic and monocytic lineages.

Patients in first remission of acute lymphoblastic leukaemia (ALL) considered to be at high risk of relapse were offered autologous bone marrow transplantation (ABMT) using purged marrow as a therapeutic alternative to cranial irradiation and maintenance chemotherapy. Twenty-seven bone marrows taken in remission, were purged using monoclonal antibodies (anti CD7 for T lineage and anti CD10 and/or anti CD19 for B lineage leukaemias) plus rabbit complement. Retrospective analysis of 19 purged marrows by immunophenotyping or immunoglobulin gene rearrangement studies demonstrated no evidence of disease. Engraftment was seen in 26 of the patients. No correlation was found between the numbers of infused nucleated cells or colony forming units-granulocyte-macrophage (CFU-GM) and subsequent engraftment kinetics. The actuarial disease-free survival (DFS) is 32% at 7 years (median follow-up 3.4 years). There were two transplant related deaths (actuarial risk 8%); the main cause of treatment failure has been disease recurrence with an overall actuarial risk of 67%; 76% for T-ALL (five of nine), 62% for common ALL (five of 10), two of five pre B and none of three patients with B-ALL. In these 27 high risk patients in vitro purging of remission marrow as part of ABMT appears not to improve patient outcome, although confirmation of this would require a randomized trial.

The effectiveness of a simple immunorosette technique for the depletion of common acute lymphatic leukemic (cALL) blasts from autologous bone marrow transplants was studied. Erythrocytes were sensitized with tetramolecular complexes consisting of rat anti-mouse IgG1 monoclonal antibodies (McAbs) that crosslink two different mouse McAbs. One of the McAbs was directed against glycophorin A, and the other was directed against marker glycoproteins of B cells and their precursors (CD9, CD10, CD19, or CD22). Immunorosettes were formed by addition of the sensitized erythrocytes to the cALL+ cells. After density-gradient separation of immunorosettes from mixtures of cALL+/terminal deoxynucleotidyl transferase-positive (TdT+) leukemic blasts and mononuclear bone marrow cells, nearly a 2-log depletion of leukemic cells was measured by flow cytometry. Clonogenic assays with two cALL+B-cell lines (Ros-17 and Nalm-16) were performed to compare the efficacy of complement-mediated cell lysis, immunorosette depletion, and a combination of both procedures. Complement-mediated cytotoxicity with the three McAbs in combination with baby rabbit complement yielded a 1- to 2-log cell kill. Immunorosette depletion resulted in a 3-log reduction of clonogenic units. Sequential application of the two methods (immunorosette depletion with CD19 McAb followed by a complement lysis with CD9 and CD10 McAbs) led to superior results in causing a 4- to 5-log purging effect. These purging procedures did not cause a loss of normal myeloid (granulocyte-macrophage colony-forming units, CFU-GM) or erythroid (erythroid burst-forming units, BFU-e) progenitors from the bone marrow. This study indicates that the combination of the two methods results in a highly efficient purging procedure for the removal of cALL+ cells from autologous bone marrow cells.