[show abstract][hide abstract] ABSTRACT: The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan-thioglycolic acid (chitosan-TGA) hydrogel and iron ions (Fe2+/ Fe3+). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r1 and r2) and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1 ± 0.02 nm, a hydrodynamic diameter of 94 ± 20 nm, a zeta potential of +21 ± 5 mV and an iron content of 3.6 ± 0.9 mg /ml. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than 3-fold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With 2-fold lower incubation iron concentration of TSS-SPIONs more than 3-fold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24 hours. The molar MR relaxivities (r2) value at 1.5 T was 3-fold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity and high MRI sensitivity seem to be promising for cell tracking.
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 03/2013; · 3.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.
PLoS ONE 01/2013; 8(6):e66909. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction.
Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NIS(pos)-hiPSCs) were established. Iodide uptake, efflux, and viability of NIS(pos)-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NIS(pos)-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of (123)I to follow donor cell survival and distribution and with the use of (99m)TC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NIS(pos)-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NIS(pos)-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected.
This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NIS(pos)-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.
[show abstract][hide abstract] ABSTRACT: The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.
[show abstract][hide abstract] ABSTRACT: Despite insights into the molecular pathways regulating hypoxia-induced gene expression, it is not known which cell types accomplish oxygen sensing during neo-vasculogenesis. We have developed a humanized mouse model of endothelial and mesenchymal progenitor co-transplantation to delineate the cellular compartments responsible for hypoxia response during vasculogenesis. Mesenchymal stem/progenitor cells (MSPCs) accumulated nuclear hypoxia-inducible transcription factor (HIF)-1α earlier and more sensitively than endothelial colony forming progenitor cells (ECFCs) in vitro and in vivo. Hypoxic ECFCs showed reduced function in vitro and underwent apoptosis within 24h in vivo when used without MSPCs. Surprisingly, only in MSPCs did pharmacologic or genetic inhibition of HIF-1α abrogate neo-vasculogenesis. HIF deletion in ECFCs caused no effect. ECFCs could be rescued from hypoxia-induced apoptosis by HIF-competent MSPCs resulting in the formation of patent perfused human vessels. Several angiogenic factors need to act in concert to partially substitute mesenchymal HIF-deficiency. Results demonstrate that ECFCs require HIF-competent vessel wall progenitors to initiate vasculogenesis in vivo and to bypass hypoxia-induced apoptosis. We describe a novel mechanistic role of MSPCs as oxygen sensors promoting vasculogenesis thus underscoring their importance for the development of advanced cellular therapies.
PLoS ONE 01/2012; 7(9):e44468. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs) are key players in vascular homeostasis and regeneration. Due to their robust proliferative potential and profound vessel-forming capacity, ECFCs are considered to represent an attractive tool for vascular regenerative medicine and a promising target for antiangiogenic tumor therapy. Here, we describe an easily applicable method for isolating ECFCs directly from unmanipulated adult human blood and an animal protein-free large-scale expansion system to generate more than 100 million functional ECFCs.
Methods in molecular biology (Clifton, N.J.) 01/2012; 879:381-7.
[show abstract][hide abstract] ABSTRACT: Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow-derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow-derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow-derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271(+) and SSEA-4(+) cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271(+) population. The differentiation potential of dermal SSEA-4(+) cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.
[show abstract][hide abstract] ABSTRACT: Clinical trials for therapeutic angiogenesis use blood- or bone marrow-derived hematopoietic cells, endothelial progenitor cells (EPC) and mesenchymal stromal cells (MSC) for vascular regeneration. Recently concerns have emerged that all three cell types could also contribute to atherosclerosis by foam cell formation. Therefore, we asked whether human myelomonocytic cells, EPC or MSC can accumulate lipid droplets (LD) and develop into foam cells.
LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the cell types. The impact of an initial pro-angiogenic induction on subsequent foam cell formation was studied to mimic relevant settings already used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to the pro-angiogenic stimulation was determined to delineate the operative mechanisms and establish a basis for interventional strategies.
Foam cells were formed by monocytes but not by EPC or MSC after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced and kinase inhibition almost abrogated intracellular LD accumulation in monocytes.
These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. Instead, EPC and MSC may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic effects during cell therapy.
[show abstract][hide abstract] ABSTRACT: The purpose of this study was to develop and characterize new surface-modified iron oxide nanoparticles demonstrating the efficiency to be internalized by human endothelial progenitor cells (EPCs) from umbilical cord blood.
Iron oxide nanoparticles were coated with polyacrylic acid-cysteine (PAA-Cys) by either in situ precipitation or postsynthesis. The nanoparticles were characterized by X-ray powder diffraction. EPCs were labeled with PAA-Cys-modified iron oxide nanoparticles or with uncoated nanoparticles. The relaxivity of uncoated and coated iron oxide nanoparticles as well as EPCs labeled with PAA-Cys-modified iron oxide were determined.
Addition of PAA-Cys increased the particle size from 10.4 to 144 and 197 nm, respectively. The X-ray powder diffraction pattern revealed that the particles consist of Fe(3)O(4) with a spinal structure. Postsynthesis coated particles showed a cellular uptake of 85% and 15.26 pg iron/cell. For both types of particles the relaxivity ratio was at least 2-fold higher than that of the gold standard Resovist(®).
The PAA-Cys coated iron oxide nanoparticles are a promising tool for labeling living cells such as stem cells for diagnostic and therapeutic application in cell-based therapies due to their high relaxivities and their easy uptake by cells.
Journal of Drug Targeting 12/2010; 19(7):562-72. · 2.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions.
Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate.
All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations.
Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion >1x10(8) ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. (3,4) This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.
[show abstract][hide abstract] ABSTRACT: The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation. To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue. Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins. This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord. All materials and procedures will be described.
[show abstract][hide abstract] ABSTRACT: Stem cell-based therapies are a promising prospect for regenerative medicine. Particularly, human multipotent mesenchymal stromal cells (MSCs) are currently in focus regarding their regenerative and immune modulating capacities. An increasing number of clinical trials investigating MSC efficiency and safety are ongoing. Ex vivo propagation of human MSCs is considered to be a prerequisite for MSC therapy. The to date standard use of fetal bovine serum in cell culture bears risks including xenoimmunization and transmission of pathogens. Alternatively, human platelet-derived growth factors have been efficiently implemented into routine MSC expansion protocols. In compliance with good manufacturing practice we established an effective time- and resource-saving procedure for MSC propagation in an animal serum-free system. Bone marrow was seeded without manipulation directly in pooled human platelet lysate (pHPL) and L-glutamine supplemented minimum essential medium without antibiotics. Clinical scale expanded MSCs were harvested already after primary culture. MSC quality, identity, purity and function were assessed according to a defined panel of release criteria and comparative genomic hybridization was used to determine genomic stability. Because various potential risks of MSCs have recently been reported, further research is required to prove efficiency and long-term safety of human MSCs for cell therapy.
Bio-medical materials and engineering 01/2009; 19(4-5):271-6. · 1.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Tissue Engineering Part C Methods 07/2008; 14(3):185-96. · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
The capacity of human platelet lysate (HPL) to replace FBS for clinical-scale MSC propagation was analyzed.
HPL could be efficiently produced from buffy coats. Multiplex analyses allowed a distinct HPL growth factor profile to be delineated. With a previously established two-step clinical-scale procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. With only 3 x 10(5) primary culture-derived MSCs, a mean of 4.36 x 10(8) HPL-MSCs (range, 3.01 x 10(8)-5.40 x 10(8)) was obtained within a single secondary 11- to 13-day culture step. Although morphologically distinct, HPL-MSCs and FBS-MSCs did not differ significantly in terms of immunophenotype, differentiation potential in vitro, and lack of tumorigenicity in nude mice in vivo.
Replacing FBS with HPL prevents bovine prion, viral, and zoonose contamination of the stem cell product. This new efficient FBS-free two-step procedure for clinical-scale MSC propagation may represent a major step toward challenging new stem cell therapies.
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
Regenerative Medicine 08/2007; 2(4):371-82. · 3.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. Disclosure of potential conflicts of interest is found at the end of this article.