Anna Jablonska

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Encapsulation of Mesenchymal Stem Cells by Bioscaffolds Protects Cell Survival and Attenuates Neuroinflammatory Reaction in Injured Brain Tissue after Transplantation

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Whartons jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the hosts immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Label-free CEST MRI Detection of Citicoline-Liposome Drug Delivery in Ischemic Stroke

ABSTRACT Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion.

Neonatal desensitization does not universally prevent xenograft rejection

1. Gray, V.E., Kukurba, K.R. & Kumar, S. Bioinformatics (2012). 2. González-Perez, A. & Lopez-Bigas, N. Am. J. Hum. Genet. 88, 440–449 (2011). 3. Adzhubei, I.A. et al. Nat. Methods 7, 248–249 (2010). 4. Tennessen, J.A. et al. Science 337, 64–69 (2012). 5. Ng, S.B. et al. Nature 461, 272–276 (2009). 6. Matthews, B.W. Biochim. Biophys. Acta 405, 442–451 (1975). efficient predictive statistical model (Supplementary Methods and Supplementary Figs. 2–5). A web server for evaluating novel variants with EvoD is available at http://barn.asu.edu/EvoD/. At ultraconserved sites, EvoD led to large reductions in the FPR: 55% for Condel and 41% for PolyPhen-2. We retrieved the population allele frequency of the neutral HumVar nSNVs at ultraconserved sites from a 5,400-exome data set4 and found that EvoD improved diagnoses across the spectrum of rare (<0.1%) to common (>5%) alleles (Fig. 1a). The balanced accuracy of EvoD was also significantly higher than that of Condel and PolyPhen-2 at ultraconserved sites (Table 1; P < 10-10). Furthermore, EvoD’s performance was consistent across ultra-, welland less-conserved sites, whereas Condel and PolyPhen-2 showed uneven performance across these classes (Table 1). For each nSNV, the EvoD statistical model also produced an impact score that reflected the degree of neutrality: most neutral = 0 and most non-neutral = 100. In an analysis of 244,272 nSNVs from the 1000 Genomes Project, we found that the population frequency of nSNVs decayed with increasing impact score (Fig. 1b). We therefore used the empirical distribution of EvoD scores to determine the statistical significance of a neutral or non-neutral diagnosis (P value) adaptively for variants at ultra-, welland less-conserved sites (Supplementary Fig. 2). Using EvoD, we analyzed nSNVs in an example set of eight personal HapMap exomes5 containing a total of 13,372 nSNVs at ultraconserved sites. Of these, 4% were predicted to be non-neutral (P < 0.05). An overwhelming majority (94%) of these non-neutral nSNVs were found in heterozygous genotypes that would neutralize the negative effects of recessive alleles. Similar results were observed for 35,367 nSNVs at welland less-conserved sites, which were also reflected in the neutrality heat maps showing the EvoD impact scores of nSNVs in heterozygous genotypes (Fig. 1c). In contrast, the fraction of homozygous nSNVs with high EvoD impact scores was much smaller at ultraconserved sites (Fig. 1c). EvoD predicted no more than one homozygous nSNV per exome to be non-neutral (P < 0.05) in ultraconserved sites, which is consistent with the fact that individuals contributing to HapMap sequencing do not suffer from any known Mendelian disease. Our results show that an evolution-aware approach to training and testing computational tools leads to better functional predictions for nSNVs, particularly at the most functionally important positions.

Imaging the DNA Alkylator Melphalan by CEST MRI: An Advanced Approach to Theranostics

Brain tumors are among the most lethal types of tumors. Therapeutic response variability and failure in patients have been attributed to several factors, including inadequate drug delivery to tumors due to the blood-brain barrier (BBB). Consequently, drug delivery strategies are being developed for the local and targeted delivery of drugs to brain tumors. These drug delivery strategies could benefit from new approaches to monitor the delivery of drugs to tumors. Here, we evaluated the feasibility of imaging 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan), a clinically used DNA alkylating agent, using chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), for theranostic applications. We evaluated the physicochemical parameters that affect melphalans CEST contrast and demonstrated the feasibility of imaging the unmodified drug by saturating its exchangeable amine protons. Melphalan generated a CEST signal despite its reactivity in an aqueous milieu. The maximum CEST signal was observed at pH 6.2. This CEST contrast trend was then used to monitor therapeutic responses to melphalan in vitro. Upon cell death, the decrease in cellular pH from ∼7.4 to ∼6.4 caused an amplification of the melphalan CEST signal. This is contrary to what has been reported for other CEST contrast agents used for imaging cell death, where a decrease in the cellular pH following cell death results in a decrease in the CEST signal. Ultimately, this method could be used to noninvasively monitor melphalan delivery to brain tumors and also to validate therapeutic responses to melphalan clinically.

A dextran-based probe for the targeted magnetic resonance imaging of tumours expressing prostate-specific membrane antigen

Safe imaging agents that are able to render the expression and distribution of cancer receptors, enzymes or other biomarkers would facilitate clinical screening of the disease. Here, we show that diamagnetic dextran particles that are coordinated to a urea-based targeting ligand for prostate-specific membrane antigen (PSMA) enable targeted magnetic resonance imaging (MRI) of the PSMA receptor. In a xenograft model of prostate cancer, micromolar concentrations of the dextran–ligand probe provided sufficient signal to specifically detect PSMA-expressing tumours via chemical exchange saturation transfer MRI. The dextran-based probe could be detected via the contrast that originated from dextran hydroxyl protons, thereby avoiding the need of chemical substitution for radioactive or metallic labelling. Because dextrans are currently used clinically, dextran-based contrast agents may help to extend receptor-targeted imaging to clinical MRI.A biodegradable probe made of dextran and a urea-based targeting ligand for prostate-specific membrane antigen enables targeted magnetic resonance imaging of tumours expressing the receptor in a xenograft mouse model of prostate cancer.

Neuroprotective Potential and Paracrine Activity of Stromal Vs. Culture-Expanded hMSC Derived from Wharton Jelly under Co-Cultured with Hippocampal Organotypic Slices

Regardless of enormous translational progress in stem cell clinical application, our knowledge about biological determinants of transplantation-related protection is still limited. In addition to adequate selection of the cell source well dedicated to a specific disease and optimal standardization of all other pre-transplant procedures, we have decided to focus more attention to the impact of culture time and environment itself on molecular properties and regenerative capacity of cell cultured in vitro. The aim of this investigation was to determine neuroprotection-linked cell phenotypic and functional changes that could spontaneously take place when freshly isolated Wharton’s jelly mesenchymal stem cell (WJ-MSC) undergo standard selection, growth, and spontaneous differentiation throughout passaging in vitro. For determining their neuroprotective potential, we used experimental model of human WJ-MSC co-culture with intact or oxygen-glucose-deprived (OGD) rat organotypic hippocampal culture (OHC). It has been shown that putative molecular mechanisms mediating regenerative interactions between WJ-MSC and OHC slices relies mainly on mesenchymal cell paracrine activity. Interestingly, it has been also found that the strongest protective effect is exerted by the co-culture with freshly isolated umbilical cord tissue fragments and by the first cohort of human mesenchymal stem cells (hMSCs) migrating out of these fragments (passage 0). Culturing of WJ-derived hMSC in well-controlled standard conditions under air atmosphere up to fourth passage caused unexpected decline of neuroprotective cell effectiveness toward OGD-OHC in the co-culture model. This further correlated with substantial changes in the WJ-MSC phenotype, profile of their paracrine activities as well as with the recipient tissue reaction evaluated by changes in the rat-specific neuroprotection-linked gene expression.

PET imaging of intra-arterial 89Zr bevacizumab in mice with and without osmotic opening of the blood-brain barrier: distinct advantage of intra-arterial delivery

Glioblastoma multiforme (GBM) is the most aggressive and common type of brain cancer. Five-year survival rates are below 12%, even with the most aggressive trimodal therapies. Poor blood–brain barrier (BBB) permeability of therapeutics is a major obstacle to efficacy. Intravenous administration of bevacizumab is the standard treatment for GBM. It has been recently demonstrated that a single intraarterial infusion of bevacizumab provides superior therapeutic outcomes in patients with recurrent GBM. Further GBM treatment benefits can be achieved through opening of the BBB before intraarterial infusion of bevacizumab. However, a rationale for intraarterial delivery and BBB opening when delivering antibodies is lacking. A method facilitating quantification of intraarterial delivery of bevacizumab is needed for more effective and personalized GBM treatment. Here, we demonstrate such a method using PET imaging of radiolabeled bevacizumab. Methods: Bevacizumab was conjugated with deferoxamine and subsequently radiolabeled with 89Zr. 89Zr-bevacizumab deferoxamine (89Zr-BVDFO) was prepared with a specific radioactivity of 81.4 ± 7.4 MBq/mg (2.2 ± 0.2 μCi/mg). Brain uptake of 89Zr-BVDFO on carotid artery and tail vein infusion with an intact BBB or with BBB opening with mannitol was initially monitored by dynamic PET, followed by whole-body PET/CT at 1 and 24 h after infusion. Th ex vivo biodistribution of 89Zr-BVDFO was also determined. Results: Intraarterial administration of 89Zr-BVDFO resulted in gradual accumulation of radioactivity in the ipsilateral hemisphere, with 9.16 ± 2.13 percentage injected dose/cm3 at the end of infusion. There was negligible signal observed in the contralateral hemisphere. BBB opening with mannitol before intraarterial infusion of 89Zr-BVDFO resulted in faster and higher uptake in the ipsilateral hemisphere (23.58 ± 4.46 percentage injected dose/cm3) and negligible uptake in the contralateral hemisphere. In contrast, intravenous infusion of 89Zr-BVDFO and subsequent BBB opening did not lead to uptake of radiotracer in the brain. The ex vivo biodistribution results validated the PET/CT studies. Conclusion: Our findings demonstrate that intraarterial delivery of bevacizumab into the brain across an osmotically opened BBB is effective, in contrast to the intravenous route.