Gerhard Lanzer

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).

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.

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.

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.

Blood donor screening for parvovirus B19 in Germany and Austria

BACKGROUND: Although the main transmission pathway of parvovirus B19 (B19) is typically via the respiratory route, several transfusion‐transmitted infections have been reported. To increase blood safety, all blood donations to our blood donor service have been screened by a B19 minipool real‐time nucleic acid testing (NAT) since April 2000. Additional customers have been screened since the summer of 2003.

Patatin-Like Phospholipase Domain-Containing Protein 3 rs738409-G in Recipients of Liver Transplants Is a Risk Factor for Graft Steatosis

BACKGROUND & AIMS The G-allele in position rs738409 of patatin-like phospholipase domain-containing protein 3 (PNPLA3) is associated with an increased hepatic concentration of triglyceride and is a risk factor for advanced liver disease. We investigated the association of donor and recipient risk alleles with the development of graft steatosis after liver transplantation. METHODS PNPLA3 genotypes were determined in 237 transplant recipients and in 255 organ donors. Macrovesicular steatosis was assessed by unenhanced computed tomography 5 years after liver transplantation in 95 patients and correlated with donor and recipient PNPLA3 genotype. RESULTS The risk allele was significantly more frequent in transplant recipients than in donors (42% vs 28%; P < .001). A prevalence of graft steatosis of 30% or greater significantly increased from 11.6% at 1 year after liver transplantation to 32.6% at 5 years after transplantation. Five years after liver transplantation, steatosis was present in 63.2% of patients homozygous for the rs738409-G allele, in 31.4% of heterozygous recipients, and in 12.0% of rs738409-CC recipients (P = .002). Donor genotypes were not associated with the development of graft steatosis. In multivariate regression analysis, recipients who carried rs738409-GG had a 13.7-fold higher risk of graft steatosis than recipients who carried rs738409-CC (P = .022), independent of recipient age, weight gain after liver transplantation, or the underlying disease. CONCLUSIONS Liver transplant recipients who carry rs738409-G in PNPLA3 are at increased risk for hepatic triglyceride accumulation, independent of the graft PNPLA3 genotype.

Adverse events and safety issues in blood donation—A comprehensive review

Although blood donation is generally safe, a variety of risks and complications exist, the most common being iron deficiency, vasovagal reactions and citrate-related events. In the last decades, extensive efforts have significantly improved recipient and product safety, but there is still great potential to optimise donor care. Many therapies in modern medicine depend on the prompt availability of blood products, therefore it is crucial to maintain a motivated and healthy donor pool in view of a limited number of healthy volunteers willing and able to give blood or blood components. We present a comprehensive review on adverse events addressing all types of blood donation including whole blood, plasma, platelet, peripheral blood stem cell, leucocyte and bone marrow donation. In addition, we outline strategies for the prevention and treatment of these events and give a blueprint for future research in this field.