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 monocytederived 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. STEM CELLS 2006;24:357-367

The use of animal-derived products during human stem cell processing bears the evident risk of xenogeneic prion, virus, or zoonose contamination. Human platelet lysate (HPL) has recently been recognized as a rich source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo stem cell manipulation. In this study we compared the gene expression profile of human multipotent mesenchymal stromal/stem cells (MSC) during ex vivo expansion for clinical applications under the aegis of either FBS or HPL. The Applied Biosystems 1700 Expression Array System was used for full genome expression profiling of MSC after a 12–14 day expansion period in a previously optimized low density expansion system. Data have been obtained from biological as well as technical replicates. A starting amount of 40μg total RNA was directly labeled and DIG-labeled cDNA was hybridized to Human Genome Survey Microarray V2.0. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software. We identified more than 300 genes that are differentially regulated upon culture of MSC in HPL compared to FBS. Biological processes specifically activated in HPL culture include mesoderm development, cell cycle control, hematopoiesis and angiogenesis which interestingly correspond to a considerable proportion of the regenerative function of MSC. In contrast, processes related to cell adhesion and adhesion-mediated signaling, cell structure, cell motility and cell communication are significantly upregulated in MSC after FBS in comparison to HPL culture. Replacing FBS with HPL not only avoids bovine prion, viral and zoonose contamination of MSC for clinical use. The tightly regulated gene expression profiles under the aegis of human growth factors and cytokines provided by HPL may even help to develop new stem cell therapy strategies.

Colony-forming units of endothelial progenitor cells (“CFU-EC”) have been introduced as a powerful biological marker for vascular function and cumulative cardiovascular risk. The precise mechanisms underlying the colony formation and their cellular composition are unclear. We hypothesized that “CFU-EC” display an immune cell function distinct from circulating endothelial progenitors. We performed detailed subtractive “CFU-EC” analyses in blood samples from 18 healthy volunteers. The impact of various blood cell types and kinetics of protein and gene expression were studied by cell sorting, flow cytometry, quantitative RT-PCR and full genome microarray analyses. “CFU-EC”-derived soluble factors were determined in multiplex cytokine measurements and tested during endothelial network formation. “CFU-EC” contained more than 99% CD45+ nucleated hematopoietic cells mainly comprising T cells and monocytes admixed with B and NK cells. Interestingly, purified T-cells plus monocytes formed “CFU-EC” clusters. The complete lack of colony formation after depletion of T cells or monocytes was contrasted by effective “CFU-EC” formation after depletion of CD34+ progenitors. Microarray analyses revealed an activation of immune function-related biological processes without changes in pathways assigned to angiogenesis. Soluble factors derived from “CFU-EC” cultures supported vascular regeneration in vitro. Unravelling “CFU-EC” formation as a result of a functional crosstalk between T cells and monocytes shifts expectations on vascular regenerative medicine. The data support a switch from a sole view on circulating endothelial progenitors towards models that favor the contribution of immune cells to vascular regeneration.

Coinfusion of hematopoietic stem cells with multipotent mesenchymal stromal/stem cells (MSC) to facilitate engraftment and immunomodulatory treatment of graft versus host disease (GvHD) are the two foremost applications of MSC therapy in hematology and oncology. Translation of promising experimental results into clinical applications has been mainly hampered by the strict dependence of MSC propagation from fetal bovine serum (FBS)-derived factors. We analyzed the capacity of different preparations of human platelet lysates (HPL) to replace FBS for clinical scale MSC production with cytomic, proteomic and genomic technology. Using a previously established two-step good manufacturing practice procedure for MSC propagation, we found that HPL can fully replace FBS as a growth factor supplement for clinical scale MSC preparation. HPL was reproducibly more efficient than FBS and supported the outgrowth of >400 million MSC from <0.4 million primary MSC (derived from <2mL bone marrow aspiration within 10 days ex vivo) within only 2 additional weeks of culture. Although morphologically distinct, HPL-MSC and FBS-MSC did not differ regarding their CD13+/29+/73+/90+/105+/146+/34−/45−/133−/HLA-AB+ immunophenotype. Multiplex analyses allowed delineating a distinct cytokine and growth factor profile. Whole transcriptome microarray analyses revealed a differential regulation of >300 genes after only one cycle of human as compared to bovine growth factor stimulation. Allogeneic HPL-MSC infusion in an intent to treat refractory GvHD was feasible and without infusion-related side effects in one patient. This new FBS-free two-step procedure for clinical scale MSC propagation will largely facilitate rational clinical testing of MSC based therapies. Replacing FBS with HPL excludes bovine prion, viral and zoonose contamination of the stem cell product. Based on this protocol a clinical trial for the treatment of steroid-refractory GvHD with HPL-MSC has been initiated.