* Velvio therapeutics aim at CNS reconstitution to renew stem cell populations in bone marrow, muscle and CNS, to reactivate normal cell functions (anti-senescence), to reactivate autophagy and normalize immune communication, to enhance stem cell friendly milieu (extracellular matrix), and to reverse scarring and fibrosis. Velvio has developed selective biomarkers to monitor regained functionalities and to bridge the time gap between therapy and recovery - these include MRI imaging, inflammatory cyto- and chemo/kines, and stem cell markers in peripheral blood. ALS (Amyotrophic Lateral Sclerosis) is a grave disease with still limited treatment options and a median survival of approx. 16 to 24 months after diagnosis. Long-time pulsed G-CSF - BM stem cell mobilization is safe 1 and highly effective 2 in ALS: Clinical pilot data in N=36 ALS patients revealed significant slowing of disease progression and significantly increased survival. Disease modelling and a biomarker panel identified and validated 15 long-time survivors with median OS of 3.8 yrs from start of treatment and a very limited disease progression of -0,12 ALS-FRS-R loss / month with G-CSF therapy in contrast to -0,9 ALS-FRS-R loss / month with best standard care. Among many other modes of action for G-CSF, it was nicely shown 3 that bone marrow cells drive central nervous system regeneration, here after radiation injury. With this impressive therapeutic potential in mind, we want to initiate a prospective pivotal adaptive clinical trial of G-CSF to achieve regulatory approval for 'early' ALS patients. The architecture of such a trial and the related expenditure will be discussed in the light of the high unmet medical need and the opportunity of using a 'repurposed' therapeutic compound employed over decades in hemato-oncology and intensive care medicine. Velvio believes that healthcare investors and regulatory authorities should not miss the opportunity for such a shortcut to establish a very efficacious new treatment option for desperate ALS patients.

Objective: Developing an integrative approach to early treatment response classification using survival modeling and bioinformatics with various biomarkers for early assessment of filgrastim (granulocyte colony stimulating factor) treatment effects in amyotrophic lateral sclerosis (ALS) patients. Filgrastim, a hematopoietic growth factor with excellent safety, routinely applied in oncology and stem cell mobilization, had shown preliminary efficacy in ALS. Methods: We conducted individualized long-term filgrastim treatment in 36 ALS patients. The PRO-ACT database, with outcome data from 23 international clinical ALS trials, served as historical control and mathematical reference for survival modeling. Imaging data as well as cytokine and cellular data from stem cell analysis were processed as biomarkers in a non-linear principal component analysis (NLPCA) to identify individual response. Results: Cox proportional hazard and matched-pair analyses revealed a significant survival benefit for filgrastim-treated patients over PRO-ACT comparators. We generated a model for survival estimation based on patients in the PRO-ACT database and then applied the model to filgrastim-treated patients. Model-identified filgrastim responders displayed less functional decline and impressively longer survival than non-responders. Multimodal biomarkers were then analyzed by PCA in the context of model-defined treatment response, allowing identification of subsequent treatment response as early as within 3 months of therapy. Strong treatment response with a median survival of 3.8 years after start of therapy was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profiles, and preserved pattern of fractional anisotropy as determined by magnetic resonance diffusion tensor imaging (DTI-MRI). Conclusion: Long-term filgrastim is safe, is well-tolerated, and has significant positive effects on disease progression and survival in a small cohort of ALS patients. Developing and applying a model-based biomarker response classification allows use of multimodal biomarker patterns in full potential. This can identify strong individual treatment responders (here: filgrastim) at a very early stage of therapy and may pave the way to an effective individualized treatment option.

Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD).Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated.Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis.Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis.Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease.

Background: There is an urgent demand for therapeutic options in patients with amyotrophic lateral sclerosis (ALS). Filgrastim (Granulocyte-Colony Stimulating Factor, G-CSF) is a hematopoietic growth factor with excellent safety and tolerability, routinely applied in oncology, intensive care, and stem cell mobilization for bone marrow transplantation. Preliminary pre-clinical and clinical evidence indicated potential efficacy in ALS. Methods: We offered ALS patients filgrastim as a new treatment approach (mostly five days per month, median duration 17 cycles, up to 76 cycles) and analyzed disease course, survival, and potential biomarkers in a total of 36 patients (mean age 52 years, CI 48-56, 29·7% female). The PRO-ACT database, which collects outcome data from 23 clinical trials performed with ALS patients worldwide, served as historical control and reference for modelling in this observational study. Findings: Long-term filgrastim treatment was safe and well tolerated. All filgrastim treated patients progressed significantly slower and survived longer compared to patients from PRO-ACT, supported by different statistical approaches. Particularly, Cox proportional hazard (filgrastim versus PRO-ACT: Risk Ratio 0·52, p=0·0052) and matched pair analyses (filgrastim versus PRO-ACT, 596 versus 373 days of survival, p<0·001) revealed a significant survival benefit for filgrastim treated patients. Strong treatment response to filgrastim with a median survival of 3·8 years (n=15/36, p<0·001) was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profile, and reduced fractional anisotropy (1·5 Tesla cerebral MRI DTI). A non-linear principal component analysis (NLPCA)-based biomarker analysis discovered individual patient responses as early as within three months of starting filgrastim treatment. Interpretation: Filgrastim constitutes a safe and powerful treatment option in ALS patients. Strong responding patients were identified by cytokines, stem cell function, and brain MRI. Treatment benefit was associated with younger age, however, also apparent in some of the older patients.Funding Statement: German Ministry of Research (GO-Bio; funding for biomarker development in ALS), donation by RB Leipzig.Declaration of Interests: UB an LA hold patents for clinical application of G-CSF in ALS, Orphan Drug Status is granted for EU and US by EMA and FDA–all within NeuroVision Pharma GmbH, Murnau, Germany. All other authors declare that they do not have any conflicts of interest. Ethics Approval Statement: The ethics committee of the University of Regensburg approved a retrospective analysis (ethics approval: 15- 101-0106 and 14-101-0011).

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule −1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of complex and still poorly understood etiology. Loss of upper and lower motoneurons results in death within few years after diagnosis. Recent studies have proposed neuroprotective and disease-slowing effects of granulocyte-colony stimulating factor (G-CSF) treatment in ALS mouse models as well as humans. In this study, six ALS patients were monitored up to 3.5 years during continuous high-dose G-CSF administration. Repetitive analyses were performed including blood count parameters, CD34+ hematopoietic stem and progenitor cell (HSPC) and colony forming cell (CFC) counts, serum cytokine levels and leukocyte telomere length. We demonstrate that continuous G-CSF therapy was well tolerated and safe resulting in only mild adverse events during the observation period. However, no mobilization of CD34+ HSPC was detected as compared to baseline values. CFC mobilization was equally low and even a decrease of myeloid precursors was observed in some patients. Assessment of telomere length within ALS patients' leukocytes revealed that G-CSF did not significantly shorten telomeres, while those of ALS patients were shorter compared to age-matched healthy controls, irrespective of G-CSF treatment. During G-CSF stimulation, TNF-alpha, CRP, IL-16, sVCAM-1, sICAM-1, Tie-2 and VEGF were significantly increased in serum whereas MCP-1 levels decreased. In conclusion, our data show that continuous G-CSF treatment fails to increase circulating CD34+ HSPC in ALS patients. Cytokine profiles revealed G-CSF-mediated immunomodulatory and proteolytic effects. Interestingly, despite intense G-CSF stimulation, telomere length was not significantly shortened.

Background: The success of clinical hematopoietic stem cell (HSC) transplants is highly dependent on the ability of HSC to home to bone marrow (BM). Although it is appreciated that hematopoietic recovery post-transplant is correlated to infused HSC number, greater understanding of homing mechanisms should allow us to identify improved transplant strategies that are not solely reliant on number but also HSC quality. Mobilized blood is the most common source of HSC for transplant and the gold standard mobilization agent, granulocyte-colony stimulating factor (G-CSF), is known to impair the homing ability of HSC. G-CSF-mobilization of HSC is known to be associated with reduced expression of key adhesion molecules and we hypothesize this to include junctional adhesion molecule A (JAM-A), a transmembrane protein enriched on long-term HSC. Currently, the specific role of JAM-A in HSC mobilization, homing and stem cell maintenance is not known. Aims: To assess the role of junctional adhesion molecule A (JAM-A) in mobilization, homing and engraftment of HSC as well as stem cell maintenance in BM. In addition, we aim to identify a G-CSF independent mobilization strategy that does not reduce expression of key adhesion receptors, thus providing a mobilized product with enhanced homing capacity. Methods: Anti-JAM-A antibody treatment of HSC followed by transplant into either C57BL/6 or NSG mice were used to assess the role of JAM-A in homing and engraftment of murine or human HSC, respectively. Mice were injected with anti-JAM-A blocking antibody for 3 consecutive days to determine whether prolonged inhibition of JAM-A in vivo leads to mobilization of HSC or alteration of HSC maintenance in BM. Mice were mobilized with G-CSF or a small molecule combination of the α4β1/α9β1 integrin antagonist “BOP” and the CXCR4 antagonist “AMD3100” and blood stem cells were comparatively assessed using 1) using single cell RNAseq 2) flow cytometric analysis of key adhesion molecules, including JAM-A, CXCR4, α4β1 and α9β1 and 3) homing and long-term transplantation. Results: JAM-A has multi-faceted roles in HSC regulation during steady state and following stem cell mobilization. Specifically, JAM-A is critical for HSC homing and engraftment post-transplant and contributes to the preferential maintenance of HSC quiescence in the endosteal niche (region closely associated with bone/BM interface). Furthermore, G-CSF dependent HSC mobilization significantly reduces JAM-A expression on HSC as a result of JAM-A cleavage by tumor necrosis factor α-converting enzyme, which is upregulated by G-CSF administration. In addition, HSC mobilized with G-CSF have significantly impaired homing potential, which is attributed to lower levels of JAM-A, CXCR4 as well as α4β1 and α9β1 integrins compared to cells mobilized using the small molecule combination of the α4β1/α9β1 integrin antagonist BOP and the CXCR4 antagonist AMD3100. Consequently, we show that a single injection of BOP plus AMD3100 for 1 hour was capable of mobilizing HSC and progenitors with significantly enhanced long-term multi-lineage engraftment potential compared to a 4-day G-CSF approach. Summary/Conclusion: Our results identify JAM-A as a critical regulator of stem and progenitor trafficking to and maintenance in BM and suggests the use of G-CSF independent mobilization strategies (e.g. BOP plus AMD3100) that do not abrogate JAM-A expression or function should result in improved clinical stem cell transplants.

Efficient homing of hematopoietic stem cells (HSC) from blood to the bone marrow (BM) following transplant is critical for achieving optimal engraftment. Indeed, HSC mobilized using the growth factor G-CSF are known to have reduced engraftment potential compared to native BM HSC, potentially due to impaired homing ability. Thus, the characterization of novel homing mechanisms will allow identification of improved mobilization strategies that are not solely reliant on HSC number but also HSC quality. In this study, we demonstrate junctional adhesion molecule A (JAM-A) has multi-faceted roles in HSC regulation during steady state and following mobilization. Specifically, JAM-A is critical for HSC homing and engraftment post-transplant and contributes to the maintenance of HSC quiescence in the endosteal niche. Furthermore, G-CSF mobilization significantly reduces JAM-A expression on HSC as a result of JAM-A cleavage by the enzyme TACE, which is upregulated following G-CSF. In addition, the significantly impaired homing potential of HSC mobilized with G-CSF is attributed to lower levels of JAM-A, CXCR4 as well as α4β1 and α9β1 integrins. In contrast, we show that HSC mobilized using the combination of the α4β1/α9β1 integrin antagonist BOP and the CXCR4 antagonist AMD3100 had significantly greater homing and long-term multi-lineage engraftment potential; in part due to higher levels of JAM-A, CXCR4 and α4β1/α9β1 integrins on mobilized cells. In addition, compared to G-CSF, which requires multiple doses over several days, mobilization with BOP plus AMD3100 occurred 1h following a single injection. Thus, our results identify JAM-A as a critical regulator of stem and progenitor trafficking to and maintenance in the BM and suggests the use of G-CSF independent mobilization regimes that do not abrogate JAM-A expression/function should result in improved stem cell transplants.

In secondary erythrocytosis, the elevated red cell count is powered by factors outside the erythroid compartment, for instance by raised erythropoietin (EPO) synthesis based on congenital defects of the oxygen-sensing pathway. The principal transcriptional regulator of EPO synthesis is endothelial PAS domain-containing protein 1 (EPAS 1). We present here the first report of a patient with erythrocytosis involving a mutation of amino acid 525 in EPAS1. The p.Asp525His mutation affects a residue that is farthermost from primary functional site Pro-531 of any of the erythrocytosis-related mutations that have been identified up to now.

Objectives: Multipotent mesenchymal stromal cells (MSCs) play a central role within the bone marrow (BM) niche, supporting hematopoiesis via soluble factors like cytokines and chemokines. In our study, we sought to investigate the effect of blocking transforming growth factor beta 1 (TGF-beta 1) and C-X-C motif chemokine 12 (CXCL12) receptor CXCR4 on acute myeloid leukemia (AML) cells in an MSC co-culture system. Methods: Human MSCs were obtained by BM aspirates and their phenotype and functional properties were confirmed in vitro. Co-cultures of AML cells on MSCs were initiated and compared to those on mouse fibroblasts (MS-5) and liquid cultures. Additionally, the effect of blocking CXCR4 and TGF-beta 1 on AML cells was tested with and without the addition of cytarabine. Results: MSCs from BM showed a typical phenotype and differentiation pattern. Co-culture of AML cells on MSCs resulted in a significantly higher proliferation capacity than on MS-5 or liquid culture. Blockade of TGF-beta 1 increased AML cell proliferation and chemosensibility, while the CXCR4 antagonist plerixafor showed anti-proliferative effects and did not change cytarabine-induced cell death compared to control. Discussion: Human MSCs are potent feeder cells, able to maintain AML cells in long-term culture. This favorable co-existence seems to be due in part to molecules important for communication within the niche. Blockade of TGF-beta 1 and CXCL12 was associated with different effects on AML cell proliferation and chemotherapy resistance. Conclusion: These findings suggest a strong supporting affinity between MSCs and AML cells within the leukemic niche, where TGF-beta 1 and CXCL12 pathways play an important role.

OBJECTIVE Aim of our study was to detect individual disease progression patterns of ALS patients and visualize them as function of extrinsic cortical curvature.BACKGROUND G-CSF may modulate clinical progression of patients with motor neuron disease. Serial follow-up MRI`s ware obtained for safety and monitoring disease progression during longterm compassionate use of G-CSF in ALS patients. Quantitative analysis of gray matter (GM) and white matter (WM) including cortical extrinsic curvature seems to represent a robust and sensitive biomarker for detecting changes of structure and GM/WM-proportion.METHODS So far, we evaluated MRI datasets of 4 out of 23 ALS patients. The mean ALS Functional Rating Scale revised (ALS-FRS-R) at start of treatment was 36.75. Patients were treated with individually adapted s.c. G-CSF with range of 150-720 MioIU/month up to 5 years, inclusive standard treatment. Monthly control visits with clinical exams and ALS-FRS-R were performed. Cranial MRI (3D high- resolution structural and diffusion weighted MRI at 1,5T) was obtained every three months. Extended and fully automated data post-processing was retrospectively analyzed in a pipeline as described by Deppe et.al. 2014. Briefly, DTI image-processing steps resulted in fractional anisotropy (FA) maps to detect microstructural alterations of WM. Surface reconstruction was applied to all structural data sets for individual quantitative analysis of GM, WM and extrinsic cortical curvature.RESULTS For each patient under longterm G-CSF treatment an individual disease pattern could be visualized. FA decline over time significantly correlated with disease course. Increase of extrinsic cortical curvature, FA decline of pyramidal tract, changes in GM and WM showed very individual patterns.CONCLUSIONS Earlier detection of individual quantitative parameters could enable to anticipate the potential future course of disease progression. Cortical curvature measurement may be considered as an additional marker for the evaluation of new ALS therapies. Disclosure: Dr. Baldaranov has nothing to disclose. Dr. Khomenko has nothing to disclose. Dr. Kobor has nothing to disclose. Dr. Johannesen has nothing to disclose. Dr. Grimm has nothing to disclose. Dr. Wirth has nothing to disclose. Dr. Bruun has nothing to disclose. Dr. Grassinger has nothing to disclose. Dr. Schuirer has nothing to disclose. Dr. Schulte-Mattler has nothing to disclose. Dr. Deppe has nothing to disclose. Dr. Bogdahn has nothing to disclose.

Objectives: Treatment development in ALS needs validated biomarkers. We initiated long-term follow up including assessment of multiple potential biomarkers in ALS patients treated with G-CSF. Here we present a retrospective analysis as a basis for further initiatives. Background: Not only pathogenic heterogeneity but also individual diversity in clinical courses are hallmarks for ALS. Measurement of pyramidal tract integrity by FA (Fractional Anisotropy, MRI) and estimation of active motor units by neurophysiology (MUNIX, e.g. hypothenar muscle) as markers of disease course and potential therapy effects are surrogate biomarkers. FA and MUNIX have already demonstrated sensitivity to disease progression in ALS patients. Inflammatory cells and cytokines are relevant co-factors of individual disease progression. Methods: Clinical ALS score (ALS-FRS-r), hematology and clinical chemistry were obtained monthly in 23 ALS patients (15 male, 8 female, mean age 51, 4 yrs.), who received G-CSF treatments up to 5 years. As further biomarkers (1) upper motor neuron integrity (DWI-MRI, FA) and (2) lower motor neuron function (MUNIX) were assessed every 3 months, as well as further cellular and inflammatory markers. Results: Disease progression (ALSFRS-r-decline) correlated significantly (p<0.0001) with loss of lower motor neurons (MUNIX-decline), and on an individual patient level, with decline in upper motor neuron integrity (FA-decline). Patients with more pronounced clinical progression (lower ALS-FRS-r) mobilized less monocytes (p=0.048) and pluripotent hematological stem cells (CD34+38-) (p=0.037) but more eosinophils (p=0.002) into peripheral blood. Time sectional separation of biomarkers may indicate individual response patterns. Conclusions: Quantitative markers for upper and lower motor neuron integrity such as FA and MUNIX as well as inflammatory cells such as monocytes, eosinophils or stem cells are promising biomarkers for ALS treatment development. Data are extremely robust: Funding for a prospective clinical trial is urgently needed. Acknowledgements: BMBF GO-Bio, BMBF MND Network Germany, PROACT-Database USA

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n= 31) and acute myeloid leukemia (n= 31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34(+) were significantly reduced at early time points in myelofibrosis patients, whereas CD34(+) CD38-and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34(+) hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.

The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α 9 β 1 /α 4 β 1 integrins with a single dose of a small molecule antagonist (BOP ( N -(benzenesulfonyl)- L -prolyl- L - O -(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ −/− model, demonstrated by a significant increase in PB CD34 + cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α 9 β 1 /α 4 β 1 within the endosteal niche. These results support using dual α 9 β 1 /α 4 β 1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.

Objective Here, we wanted to test the hypothesis in human neuronal progenitor cells whether antisense-oligonucleotide (ASO)-mediated inhibition of TGF-β signaling will allow regeneration of adult neurogenesis. Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no effective treatment so far. The current molecular genetic campaign is increasingly elucidating the molecular pathogenesis of this fatal disease; previous studies demonstrate high concentrations of Transforming Growth Factor-β (TGF-β) in Cerebrospinal Fluid (CSF) of traumatic-brain-injury patients. These high levels of circulating TGF-β are known to promote stem cell quiescence and therefore lead to the inhibition of adult neurogenesis within the subventricular zone (SVZ) of the brain. Thus, the local reduction of TGF- β signaling might promote neuronal regeneration. Methods First we investigated if the immortalized human neuronal progenitor ReNcell CX (Millipore) represents a reliable model to analyze TGF-β-mediated effects: we first examined the ability of ReNcell CX to respond to TGF-β by studying respective pathway molecules with qRT-PCR, immunoblotting and immunocytochemistry. Next, to monitor whether TGF-β1 induces cell cycle quiescence, ReNcell CX cells were treated with TGF-β1 (10 and 50 ng/ml) for 7 days followed by a 96-h incubation with a TGF-βRII-ASO (10 µM). The effectivity of the ASO treatment on reducing TGF-β signaling, and blocking TGF-β-mediated effects on cell cycle quiescence was investigated via qRT-PCR, immunoblotting, and immunocytochemistry. Results We could demonstrate that blocking of TGF-β signaling in ReNcell CX cells by selective ASO`s lead to reactivated adult neurogenesis. Gymnotic TGF-βRII-ASO transfer downregulates the target TGF-βRII, resulting in an inhibited TGF-β-signaling in ReNcell CX cells. Increased proliferation of precursor cells was documented, paralleled by increased levels of neuronal markers like NeuN. Conclusion The ReNcell CX cells represent an adequate model to investigate TGF-β signaling-mediated effects on neuronal precursor cells and on their reactivation for increased neurogenesis by a TGF-βRII-ASO.