Wolfgang Böcker

Introducing a single‐cell‐derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence‐associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single‐cell‐derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chon‐drogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

IKK-2 is required for TNF-α-induced invasion and proliferation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-κB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-α (TNF-α), which is known to be a key regulator of the NF-κB pathway. Therefore, we hypothesized that TNF-α may lead to MSC invasion and proliferation by activation of the NF-κB pathway. TNF-receptor 1 and 2, NF-κB (p65), and IκB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-α caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-α strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-κB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-α stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-κB pathway caused a significant reduction in the basal proliferation rate. TNF-α stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-α led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-κB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-α-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-κB signal transduction pathway.

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self‐renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms.

Vascular Incorporation of Endothelial Colony-Forming Cells Is Essential for Functional Recovery of Murine Ischemic Tissue Following Cell Therapy

Objective—Cord blood–derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell–based therapy, including mesenchymal stem cells (MSCs). Methods and Results—ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor+ cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. Conclusion—Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell–based therapy represents a promising approach for vascular regeneration of ischemic tissue.

Diagnostic Value of the Glasgow Coma Scale for Traumatic Brain Injury in 18,002 Patients with Severe Multiple Injuries

Although patients with severe multiple injuries may have other reasons for unconsciousness, traumatic brain injury (TBI) in these patients is frequently defined by the Glasgow Coma Scale (GCS). Nevertheless, the diagnostic value of GCS for severe TBI in the multiple-injured patient is unknown. Therefore, we investigated the diagnostic value of GCS to identify severe TBI in multiple-injured patients. The records of 18,002 severely injured adult (ISS >16) patients from the Trauma Register of the German Society for Trauma Surgery were analyzed and initial GCS and Abbreviated Injury Scale (head) (AIS(head)) were recorded. A severe TBI was defined by an AIS(head) ≥ 3. On the other hand, unconsciousness was defined by an initial GCS ≤ 8. By these criteria, 6546 patients (36.3%) were unconscious, and 8746 patients (48.6%) had severe TBI. Nine percent of all cases (n=1643) had a GCS ≤ 8 without severe TBI. Only 56.1% of patients with severe TBI (n=4903) had been unconscious. Decreasing levels of unconsciousness (as defined by GCS) showed consistent rising prevalence of severe TBI (correlation coefficient r=-0.52). Approximately 20% of all multiple-injured patients arriving in the emergency department with an initial GCS of 15 had severe TBI (AIS(head) ≥ 3). The diagnostic value of GCS ≤ 8 for severe TBI in patients with multiple injuries has low sensitivity (56.1%) but higher specificity (82.2%). Our study indicates that the GCS (as defined ≤ 8) in unconsciousness patients with multiple injuries shows only a moderate correlation with the diagnosis of severe TBI. Nevertheless, the main reason for unconsciousness in patients with multiple injuries is TBI, since only 9% of these patients had another reason for unconsciousness. However, due to the poor sensitivity of GCS, we suggest the use of the anatomical scoring system with AIS(head) ≥ 3 to define severe TBI in patients with multiple injuries.

First inducible transgene expression in porcine large animal models.

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet‐on system. This expression system is activated by doxycycline (dox) via the tet‐controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken β‐actin promoter (CAG). Then, cells from CAG‐TA transgenic founders were nucleofected with TRE‐controlled expression vectors for either porcine cytotoxic T‐lymphocyte associated antigen 4‐Fc domain of immunoglobulin G1 (CTLA‐4Ig) or soluble receptor activator of NF‐κB ligand (RANKL), and double‐transgenic offspring were cloned. Dox administration resulted in a dose‐dependent increase in expression of CTLA‐4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double‐transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG‐TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.—Klymiuk, N., Böcker, W., Schönitzer, V., Bahr, A., Radic, T., Fröhlich, T., Wünsch, A., Keßler, B., Kurome, M., Schilling, E., Herbach, N., Wanke, R., Nagashima, H., Mutschler, W., Arnold, G. J., Schwinzer, R., Schieker, M., Wolf, E. First inducible transgene expression in porcine large animal models. FASEB J. 26, 1086‐1099 (2012). www.fasebj.org

Human mesenchymal stem cells at the single-cell level: simultaneous seven-colour immunofluorescence

Extracellular, intracellular or surface proteins can be used as putative markers to characterize human mesenchymal stem cells (hMSC). However, these markers are also expressed by other cell types and primary cell pools reveal considerable heterogeneity. Therefore, the simultaneous detection of several markers on a single cell appears to be an attractive approach to identify hMSC. Here we demonstrate the specific distinction of human MSC from human osteoblasts via seven‐colour fluorescence on the single cell level with simultaneous marker detection of CD44, CD105/endoglin, CD106/VCAM‐1, collagen‐IV, fibronectin, actin and DAPI nuclear staining. We performed spectral image acquisition using a Sagnac‐type interferometer. Subsequent linear unmixing allowed for decomposition of each pixel in its spectral components. Our approach reveals a typical expression profile of the adherent singular cells, allowing the specific distinction between hMSC and osteoblasts on the single cell level.

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression. HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and β1. HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients. In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients.

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long‐term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time‐consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs.

Implications of combined ovariectomy and glucocorticoid (dexamethasone) treatment on mineral, microarchitectural, biomechanical and matrix properties of rat bone

Osteoporosis is one of the deleterious side effects of long‐term glucocorticoid therapy. Since the condition is particularly aggressive in postmenopausal women who are on steroid therapy, in this study we have attempted to analyse the combined effect of glucocorticoid (dexamethasone) treatment and cessation of oestrogen on rat bone. The dual aim was to generate osteoporotic bone status in a short time scale and to characterise the combination of glucocorticoid–postmenopausal osteoporotic conditions. Sprague Dawley rats (N = 42) were grouped randomly into three groups: untreated control, sham‐operated and ovariectomized–steroid (OVX‐Steroid) rats. Control animals were euthanized with no treatment [Month 0 (M0)], while sham and OVX‐Steroid rats were monitored up to 1 month (M1) and 3 months (M3) post laparotomy/post OVX‐Steroid treatment. Histology, dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (micro‐CT), and biomechanical and mRNA expression analysis of collagenous, non‐collagenous matrix proteins and osteoclast markers were examined. The study indicated enhanced osteoclastogenesis and significantly lower bone mineral density (BMD) in the OVX‐Steroid rats with Z‐scores below −2.5, reduced torsional strength, reduced bone volume (BV/TV%), significantly enhanced trabecular separation (Tb.S), and less trabecular number (Tb.N) compared with sham rats. Osteoclast markers, cathepsin K and MMP 9 were upregulated along with Col1α1 and biglycan with no significant expression variation in fibronectin, MMP 14, LRP‐5, Car II and TNC. These results show higher bone turnover with enhanced bone resorption accompanied with reduced torsional strength in OVX‐Steroid rats; and these changes were attained within a short timeframe. This could be a useful model which mimics human postmenopausal osteoporosis that is associated with steroid therapy and could prove of value both in disease diagnosis and for testing generating and testing biological agents which could be used in treatment.