Erik Pittermann

Is the intravascular administration of mesenchymal stem cells safe?: Mesenchymal stem cells and intravital microscopy

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n= 11–14, P< 0.05) and decreased right ventricular wall stress (n= 8, P< 0.05) assessed by pressure‐volume loops after 6 weeks. MI‐EPO hearts exhibited smaller infarction size (20.1 ± 1.1%versus 27.8 ± 1.2%; n= 6–8, P< 0.001) and greater capillary density (338.5 ± 14.7 versus 259.8 ± 9.2 vessels per mm; n= 6–8, P< 0.001) than MIC hearts. Direct EPO injection reduced post‐MI myocardial apoptosis by approximately 41% (0.27 ± 0.03%versus 0.42 ± 0.03%; n= 6, P= 0.005). The chemoattractant SDF‐1 was up‐regulated significantly assessed by quantitative realtime PCR and immunohistology. c‐Kit+ and CD34+ stem cells were significantly more numerous in MI‐EPO than in MIC at 24 hrs in peripheral blood (n= 7, P< 0.05) and 48 hrs in the infarcted hearts (n= 6, P< 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI‐EPO hearts (n= 7, P< 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c‐Kit+ and CD34+ stem cells via the enhanced expression of chemoattractant SDF‐1.

A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantation into the injured heart.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells characterized by their self-renewal and differentiation potential. Accumulating clinical and preclinical evidence indicate MSCs are a promising cell source for regenerative medical therapies. However, undesirable immortalization, spontaneous transformation, and tumorigenic potential from long-term cultured MSCs have been reported in human and mouse. We report rat MSCs isolated from young donors could undergo transformation in early passage culture. We aimed to characterize the transformed population and determine their therapeutic effects after intracardiac transplantation in the infarcted myocardium. MSCs were isolated from bone marrow of Lewis rats according to standard protocols and cultured under standard conditions. Phenotype of growing cells was assessed by flow cytometry. Following acute myocardial infarction in rats, cells were delivered by intracardiac injection. Cardiac functions were assessed by pressure–volume loops. Infarction size and pathologic effects were evaluated after 6 weeks. The abnormal colonies were detected in culture as early at passage 3. They were noted to appear as distinctly different morphology from typical MSCs, which changed from a normal elongated spindle shape to a compact abnormal morphology. They exhibited rapid cell proliferation. Some subclones lost contact inhibition of cell division and formed multilayer aggregates. Chromosomal instability was detected. They were devoid of surface markers CD29, CD44, CD90, and CD117. Furthermore, there was no significant improvement on infarction size and cardiac function 6 weeks after cell transplantation. Our study highlights the need for establishment of biosafety criteria in regulating culture-expanded MSCs to achieve the full clinical therapeutic benefits.

One Step Creation of Multifunctional 3D Architectured Hydrogels Inducing Bone Regeneration

Structured hydrogels showing form stability and elastic properties individually tailorable on different length scales are accessible in a one-step process. They support cell adhesion and differentiation and display growing pore size during degradation. In vivo experiments demonstrate their efficacy in biomaterial-induced bone regeneration, not requiring addition of cells or growth factors.

Human Mesenchymal Stem Cells Display Reduced Expression of CD105 after Culture in Serum-Free Medium

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However, ex vivo expansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.

Viability of human mesenchymal stem cells seeded on crosslinked entropy-elastic gelatin-based hydrogels.

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.

Intracardiac Erythropoietin Injection Reveals Antiinflammatory Potential and Improved Cardiac Functions Detected by Forced Swim Test

Systemic administration of erythropoietin (Epo) protects the myocardium from an ischemic insult and promotes beneficial remodeling. We hypothesized that intracardiac injection of Epo may exhibit cardioprotective potential with reduced systemic toxicity. Following myocardial infarction (MI), Epo was injected directly into the border of the infarction. Six weeks after an MI, we evaluated infarction size, angiogenesis, and pathologic effects of the treatment. Myocardial performance was assessed with a Forced Swim Test adapted to the study. Anti-inflammatory and cellular proliferative effects of Epo were analyzed by measuring expression of integrin-beta and CdK4 by reverse transcriptase-polymerase chain reaction (RT-PCR). The findings indicated improved cardiac status with direct Epo administration. Exercise capacity detected by the Forced Swim Test was significantly increased. There was radical reduction of absolute infarction size, ventricular dilatation, and hypertrophy in the Epo group. Integrin-beta was down-regulated and CdK4 expression was increased significantly with Epo. In conclusion, the study demonstrated that intramyocardial Epo injection, following MI, reduced inflammation, enhanced angiogenesis and proliferation, improved myocardial functions, and did not lead to intramural thrombus formation.

HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.

High‐mobility group box 1 (HMGB‐1) is a strong chemo‐attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB‐1–mediated adhesion and rolling of c‐kit+ cells and assess whether toll‐like receptor‐2 (TLR‐2) and toll‐like receptor‐4 (TLR‐4) of endothelial cells or c‐kit+ cells are implicated in the activation of downstream migration signals to peripheral c‐kit+ cells. Effects of HMGB‐1 on the c‐kit+ cells/endothelial interaction were evaluated by a cremaster muscle model in wild‐type (WT), TLR‐2 (−/−) and Tlr4 (LPS‐del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real‐time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB‐1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c‐kit+ cells was more than 13‐fold higher than that of the control group (14.6 ± 5.1 cells/mm2 in ‘WT’ versus 1.1 ± 1.0 cells/mm2 in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR‐2 (−/−) and Tlr4 (LPS‐del) mice compared to WT mice (1.5 ± 1.4 cells/mm2 in ‘TLR‐2 (−/−)’ and 2.4 ± 1.4 cells/mm2 in ‘Tlr4 (LPS‐del)’ versus 14.6 ± 5.1 cells/mm2 in ‘WT’, P < 0.05). TLR‐2 (−/−) and Tlr4 (LPS‐del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB‐1 mediates c‐kit+ cell recruitment via endothelial TLR‐2 and TLR‐4.

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross‐clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross‐clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34+ cells and the expression of brain‐derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin‐treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline‐treated mice with complete paralysis. Motor neurons in erythropoietin‐treated mice were more preserved at day 7 than those in saline‐treated mice with complete paralysis. CD34+ cells in the lumbar spinal cord of erythropoietin‐treated mice were more abundant at day 2 than those of saline‐treated mice. Brain‐derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin‐treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34+ cells, and enhanced expression of brain‐derived neurotrophic factor and vascular endothelial growth factor.

Macromol. Biosci. 3/2016

Back Cover: Defined poly(2-oxazoline)s (POx) bearing a maleimide terminal function was conjugated with hydrophilic elastin-like polypeptides (ELP) in a straightforward Michael-addition reaction. The first example to use the amphiphilic POx-ELP hybrids as a polymer therapeutic by the formulation of a hydrophobic anti-cancer drug (paclitaxel) is shown. Further details can be found in the article by J. F. Nawroth, J. R. McDaniel, A. Chilkoti, R. Jordan, R. Luxenhofer on page 322.