New strategies in hematopoietic stem cell transplantation: G-CSF-mobilized unprocessed whole blood

Transplantation of mobilized peripheral blood stem cells (PBSC) for rescue of bone marrow function after high-dose chemo-/radiotherapy is widely used in hematologic malignancies and solid tumors. Mobilization of stem cells to the peripheral blood can be achieved by cytokine treatment of the patients. The main advantage of autologous PBSC transplantation over bone marrow transplantation is the faster recovery of neutrophil and platelet counts. The threshold number of PBSC required for adequate rescue of bone marrow is thought to be about 2 x 106 CD34+ cells/kg, if the stem cells are collected by leukapheresis and subsequently cryopreserved. We show that this critical number could be further reduced to as few as 0.2 x 106 cells/kg. In 30 patients with multiple myeloma and 25 patients with bad risk lymphoma 1 liter of granulocyte colony-stimulating factor (G-CSF)-mobilized unprocessed whole blood (stored at 4oC for 1-3 days) was used for transplantation. Compared to a historical control group, a significant reduction in the duration of neutropenia, thrombocytopenia and the length of hospital stay was documented. Furthermore, the effect of stem cell support was reflected by a lower need for platelet and red cell transfusions and a reduced antibiotic use. Considering the data as a whole, a cost saving of about 50% was achieved. To date, this easy to perform method of transplantation is only feasible following high-dose therapies that are completed within 72 h, since longer storage of unprocessed blood is accompanied by a substantial loss of progenitor cell function. Ongoing investigations include attempts to prolong storage times for whole bloo

The phenotypic profile of CD34-positive peripheral blood stem cells in different mobilization regimens

The type of regimen used might result in mobilization of phenotypically and functionally different CD34+ cells. We compared the phenotype of CD34+ cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony-stimulating factor (G-CSF) alone (n = 13); II, patients mobilized with G-CSF following chemotherapy (n = 16); and III, patients mobilized with G-CSF after high-dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34+ subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L-selectin expression. Regimens I and II led to higher numbers of mobilized CD34+ cells (mean 468 x 106 and 491 x 106 CD341 cells per leukapheresis procedure respectively) than regimen III (mean 41 x 106 CD34+ cells per leukapheresis procedure). Both the expression of L-selectin and CD54 on CD34+ cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41+) progenitors. A higher percentage of primitive (CD38- and/or HLA=DR=) CD34+ cells was found in group III, correlating with a higher clonogenicity of the CD34+ cells. However, when comparing the CD34+ subpopulations that were also positive for L-selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5.1% of CD341 being CD41+/L-selectin+ compared with 9.2% and 8.9% in regimens I and II respectively. We concluded that the phenotypes of the CD34+ cells in the G-CSF (group I) and G-CSF-chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34+ cells mobilized in the high-dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34+ cells with a phenotypically favourable phenotype

Homing and clonogenic outgrowth of CD34+ peripheral blood stem cells: A role for L-selectin?

Objective. After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin + stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. Materials and Methods. Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34+ cells with or without blocking of L-selectin–ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin+ L-selectin- cells or in the presence of antibodies. Results. Blocking of L-selectin on CD34+ cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti–L-selectin monoclonal antibody-incubated CD34+ cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. Conclusion.Using in vitro assays for CD34+ stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

Stem cell doses necessary for engraftment after myeloablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8– 3.0 x 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 x 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3– 0.4 x 106 CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4ºC, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery

Early Apoptosis Largely Accounts for Functional Impairment of CD34+ Cells in Frozen–Thawed Stem Cell Grafts

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34+ enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34+ cells, using the vital stain Syto16. Sytohigh/7-AAD2 cells were defined as viable, Syto16low/7-AAD2 cells as early apoptotic and Syto16low/7-AAD1 as dead. This was confirmed in a subsequent study using frozen–thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34+ cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4°C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26–33%) after cryopreservation matched CD34+ recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34+ cells had lost migratory ability toward stromal cell derived factor-1a (SDF-1a). The establishment of a Syto16high/7-AAD2 proportion of CD34+ cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34+ cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34+ assessment of post-thawed samples in clinical flow cytometry laboratories

Homing and clonogenic outgrowth of CD34+ peripheral blood stem cells: A role for L-selectin?

Objective. After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin+ stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. Materials and Methods. Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34+ cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin+L-selectin- cells or in the presence of antibodies. Results. Blocking of L-selectin on CD34+ cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34+ cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. Conclusion. Using in vitro assays for CD34+ stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc

Data sharing under the general data protection regulation: Time to harmonize law and research ethics?

The General Data Protection Regulation (GDPR) became binding law in the European Union Member States in 2018, as a step toward harmonizing personal data protection legislation in the European Union. The Regulation governs almost all types of personal data processing, hence, also, those pertaining to biomedical research. The purpose of this article is to highlight the main practical issues related to data and biological sample sharing that biomedical researchers face regularly, and to specify how these are addressed in the context of GDPR, after consulting with ethics/legal experts. We identify areas in which clarifications of the GDPR are needed, particularly those related to consent requirements by study participants. Amendments should target the following: (1) restricting exceptions based on national laws and increasing harmonization, (2) confirming the concept of broad consent, and (3) defining a roadmap for secondary use of data. These changes will be achieved by acknowledged learned societies in the field taking the lead in preparing a document giving guidance for the optimal interpretation of the GDPR, which will be finalized following a period of commenting by a broad multistakeholder audience. In parallel, promoting engagement and education of the public in the relevant issues (such as different consent types or residual risk for re-identification), on both local/national and international levels, is considered critical for advancement. We hope that this article will open this broad discussion involving all major stakeholders, toward optimizing the GDPR and allowing a harmonized transnational research approach. © 2021 Lippincott Williams and Wilkins. All rights reserved