Werner Linkesch

Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.

BACKGROUND: Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodulatory cellular therapies. Translation of auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS).

Standard graft-versus-host disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial

BACKGROUND Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic haematopoietic cell transplantation from unrelated donors. Anti-T-cell globulins (ATGs) might lower the incidence of GVHD. We did a prospective, randomised, multicentre, open-label, phase 3 trial to compare standard GVHD prophylaxis with ciclosporin and methotrexate with or without anti-Jurkat ATG-Fresenius (ATG-F). METHODS Between May 26, 2003, and Feb 8, 2007, 202 patients with haematological malignancies were centrally randomly assigned using computer-generated centre-stratified block randomisation between treatment groups receiving ciclosporin and methotrexate with or without additional ATG-F. One patient in the ATG-F group did not undergo transplantation, thus 201 patients who underwent transplantation with peripheral blood (n=164; 82%) or bone marrow (n=37; 18%) grafts from unrelated donors after myeloablative conditioning were included in the full analysis set, and were analysed according to their randomly assigned treatment (ATG-F n=103, control n=98). The primary endpoint was severe acute GVHD (aGVHD) grade III-IV or death within 100 days of transplantation. The trial is registered with the numbers DRKS00000002 and NCT00655343. FINDINGS The number of patients in the ATG-F group who had severe aGVHD grade III-IV or who died within 100 days of transplantation was 12 and 10 (21.4%, 95% CI 13.4-29.3), respectively, compared with 24 and nine (33.7%, 24.3-43.0) patients, respectively, in the control group (adjusted odds ratio 0.59, 95% CI 0.30-1.17; p=0.13). The cumulative incidence of aGVHD grade III-IV was 11.7% (95% CI 6.8-19.8) in the ATG-F group versus 24.5% (17.3-34.7) in the control group (adjusted hazard ratio [HR] 0.50, 95% CI 0.25-1.01; p=0.054), and cumulative incidence of aGVHD grade II-IV was 33.0% (n=34; 95% CI 25.1-43.5) in the ATG-F group versus 51.0% (n=50; 95% CI 42.0-61.9) in the control group (adjusted HR 0.56, 0.36-0.87; p=0.011). The 2-year cumulative incidence of extensive chronic GVHD was 12.2% (n=11; 95% CI 7.0-21.3) versus 42.6% (n=34; 95% CI 33.0-55.0; adjusted HR 0.22, 0.11-0.43; p<0.0001). There were no differences between treatment groups with regard to relapse, non-relapse mortality, overall survival, and mortality from infectious causes. INTERPRETATION The addition of ATG-F to GVHD prophylaxis with ciclosporin and methotrexate resulted in decreased incidence of acute and chronic GVHD without an increase in relapse or non-relapse mortality, and without compromising overall survival. The use of ATG-F is safe for patients who are going to receive a haematopoietic cell transplantation from matched unrelated donors. FUNDING Fresenius Biotech GmbH.

Blood Monocytes Mimic Endothelial Progenitor Cells

The generation of endothelial progenitor cells (EPCs) from blood monocytes has been propagated as a novel approach in the diagnosis and treatment of cardiovascular diseases. Low‐density lipoprotein (LDL) uptake and lectin binding together with endothelial marker expression are commonly used to define these EPCs. Considerable controversy exists regarding their nature, in particular, because myelomonocytic cells share several properties with endothelial cells (ECs). This study was performed to elucidate whether the commonly used endothelial marker determination is sufficient to distinguish supposed EPCs from monocytes. We measured endothelial, hematopoietic, and progenitor cell marker expression of monocytes before and after angiogenic culture by fluorescence microscopy, flow cytometry, and real‐time reverse transcription–polymerase chain reaction. The function of primary monocytes and monocyte‐derived supposed EPCs was investigated during vascular network formation and EC colony‐forming unit (CFU‐EC) development. Monocytes cultured for 4 to 6 days under angiogenic conditions lost CD14/CD45 and displayed a commonly accepted EPC phenotype, including LDL uptake and lectin binding, CD31/CD105/CD144 reactivity, and formation of cord‐like structures. Strikingly, primary monocytes already expressed most tested endothelial genes and proteins at even higher levels than their supposed EPC progeny. Neither fresh nor cultured monocytes formed vascular networks, but CFU‐EC formation was strictly dependent on monocyte presence. LDL uptake, lectin binding, and CD31/CD105/CD144 expression are inherent features of monocytes, making them phenotypically indistinguishable from putative EPCs. Consequently, monocytes and their progeny can phenotypically mimic EPCs in various experimental models.

Chronic graft-versus-host disease: long-term results from a randomized trial on graft-versus-host disease prophylaxis with or without anti-T-cell globulin ATG-Fresenius

Previous randomized graft-versus-host disease (GVHD)-prophylaxis trials have failed to demonstrate reduced incidence and severity of chronic GVHD (cGVHD). Here we reanalyzed and updated a randomized phase 3 trial comparing standard GVHD prophylaxis with or without pretransplantation ATG-Fresenius (ATG-F) in 201 adult patients receiving myeloablative conditioning before transplantation from unrelated donors. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (P < .0001). The 3-year cumulative incidence of relapse and of nonrelapse mortality was 32.6% and 19.4% in the ATG-F group and 28.2% and 33.5% in the control group (hazard ratio [HR] = 1.21, P = .47, and HR = 0.68, P = .18), respectively. This nonsignificant reduction in nonrelapse mortality without increased relapse risk led to an overall survival rate after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, P = .39, nonsignificant). The HR for receiving immunosuppressive therapy (IST) was 0.31 after ATG-F (P < .0001), and the 3-year probability of survival free of IST was 52.9% and 16.9% in the ATG-F versus control, respectively. The addition of ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis lowers the incidence and severity of cGVHD, and the risk of receiving IST without raising the relapse rate. ATG-F prophylaxis reduces cGVHD morbidity.

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Immune Cells Mimic the Morphology of Endothelial Progenitor Colonies In Vitro

Endothelial progenitor cells (EPC) are considered powerful biologic markers for vascular function and cardiovascular risk, predicting events and death from cardiovascular causes. Colony‐forming units of endothelial progenitor cells (CFU‐EC) are used to quantify EPC circulating in human peripheral blood. The mechanisms underlying colony formation and the nature of the contributing cells are not clear. We performed subtractive CFU‐EC analyses to determine the impact of various blood cell types and kinetics of protein and gene expression during colony formation. We found that CFU‐EC mainly comprise T cells and monocytes admixed with B cells and natural killer cells. The combination of purified T cells and monocytes formed CFU‐EC structures. The lack of colonies after depletion or functional ablation of T cells or monocytes was contrasted with effective CFU‐EC formation in the absence of CD34+ cells. Microarray analyses revealed activation of immune function‐related biological processes without changes in angiogenesis‐related processes during colony formation. In concordance with a regenerative function, soluble factors derived from CFU‐EC cultures supported vascular network formation in vitro. Recognizing CFU‐EC formation as the result of a functional cross between T cells and monocytes shifts expectations of vascular regenerative medicine. Our data support the move from a view of circulating EPC toward models that include a role for immune cells in vascular regeneration.

Rapid Large-Scale Expansion of Functional Mesenchymal Stem Cells from Unmanipulated Bone Marrow Without Animal Serum

Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of > 2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.

Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application

BACKGROUND Umbilical cord blood (UCB) is an easily accessible alternative source for multipotent mesenchymal stromal cells (MSCs) and is generally believed to provide MSCs with a higher proliferative potential compared with adult bone marrow. Limitations in cell number and strict dependence of expansion procedures from selected lots of fetal bovine serum have hampered the progress of clinical applications with UCB-derived MSCs. METHODS We analyzed the isolation and proliferative potential of human UCB MSCs compared with bone marrow MSCs under optimized ex vivo culture conditions. We further investigated human platelet lysate as an alternative to replace fetal bovine serum for clinical-scale MSC expansion. Clonogenicity was determined in colony-forming units-fibroblast assays. MSC functions were tested in hematopoiesis support, vascular-like network formation and immune modulation potency assays. RESULTS MSCs could be propagated from UCB with and without fetal bovine serum. MSC propagation was effective in 46% of UCB samples. Once established, the proliferation kinetics of UCB MSCs did not differ significantly from that of bone marrow MSCs under optimized culture conditions, resulting in more than 50 population doublings after 15 weeks. A clinical quantity of 100 million MSCs with retained differentiation potential could be obtained from UCB MSCs within approximately 7 weeks. Ex vivo expansion of hematopoietic UCB-derived CD34+ cells as well as immune inhibition and vascular-like network formation could be shown for UCB MSCs propagated under both culture conditions. CONCLUSION We demonstrate for the first time that human MSCs can be obtained and propagated to a clinical quantity from UCB in a completely bovine serum-free system. Surprisingly, our data argue against a generally superior proliferative potential of UCB MSCs. Functional data indicate the applicability of clinical-grade UCB MSCs propagated with human platelet lysate-conditioned medium for hematopoiesis support, immune regulation and vascular regeneration.

Treatment of Extranodal Marginal Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue Type With Cladribine: A Phase II Study

PURPOSE As chemotherapy has not been extensively studied in patients with lymphoma of the mucosa-associated lymphoid tissue (MALT), we initiated a prospective study to evaluate the activity of the nucleoside analog cladribine (2-chlorodeoxyadenosine [2-CdA]) in this disease. PATIENTS AND METHODS Patients with histologically verified MALT-type lymphoma were enrolled. 2-CdA was administered at a dose of 0.12 mg/kg body weight on 5 consecutive days, as a 2-hour infusion. Cycles were repeated every 4 weeks for a maximum of six cycles. RESULTS Nineteen patients with gastric and seven patients with extragastric MALT lymphoma were enrolled. All patients were chemotherapy-naive, and two had been locally irradiated before systemic relapse of the lymphoma. A total of 102 cycles was administered to our patients (median number of cycles per patient, four). All 25 assessable patients responded to treatment: 21 patients (84%) achieved complete remission (CR) and four patients achieved partial remission. All patients (100%) with gastric presentation, but only three patients (43%) with extragastric presentation, achieved CR. Toxicities were moderate and mainly hematologic and required dose reduction and/or premature discontinuation of therapy in only three cases. Two patients died from vascular events, one shortly after the first cycle because of myocardial infarction and the other from stroke 3 months after the second course. Three patients relapsed after 13, 18, and 22 months and one patient showed progressive disease after 15 months. At present, 24 patients are alive at a median follow-up time of 32 months. CONCLUSION Our data demonstrate that 2-CdA is highly effective in inducing CR in 84% of patients with MALT-type lymphoma.

Two steps to functional mesenchymal stromal cells for clinical application

BACKGROUND: Ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) is a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Even large volumes of starting material and extended culture periods, however, do not necessarily produce 2 × 106 MSCs per kg per adult patient. A new two‐step procedure has been devised to propagate more than 1 × 108 MSCs from small marrow volumes within fewer than 4 weeks.