Publications

Perineuronal Nets and Their Role in Synaptic Homeostasis

Mateusz Bosiacki 1, Magdalena Ga˛ssowska-Dobrowolska 2, Klaudyna Kojder 3,
Marta Fabianska 4, Dariusz Jezewski 5, Izabela Gutowska 6 and Anna Lubkowska 1,
1 Department of Functional Diagnostics and Physical Medicine, Pomeranian Medical University in Szczecin,Zołnierska 54 Str., 71-210 Szczecin, Poland
2 Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences,Pawinskiego 5 Str., 02-106Warsaw, Poland
3 Department of Anaesthesiology and Intensive Care, Pomeranian Medical University in Szczecin,71-252 Szczecin, Poland
4 Institute of Philosophy, University of Szczecin, Krakowska 71-79 Str., 71-017 Szczecin, Poland
5 Department of Neurosurgery and Pediatric Neurosurgery, Department of Applied Neurocognitivistics, Pomeranian Medical University in Szczecin, 71-252 Szczecin, Poland
6 Department of Human Nutrition and Metabolomics, Department of Medical Chemistry, Pomeranian Medical University in Szczecin, Broniewskiego 24 Str., 71-252 Szczecin, Poland

Received: 7 July 2019; Accepted: 16 August 2019; Published: 22 August 2019


Abstract: Extracellular matrix (ECM) molecules that are released by neurons and glial cells form perineuronal nets (PNNs) and modulate many neuronal and glial functions. PNNs, whose structure is still not known in detail, surround cell bodies and dendrites, which leaves free space for synapses to come into contact. A reduction in the expression of many neuronal ECM components adversely a ects processes that are associated with synaptic plasticity, learning, and memory. At the same time, increasedECMactivity, e.g., as a result of astrogliosis following brain damage or in neuroinflammation, can also have harmful consequences. The therapeutic use of enzymes to attenuate elevated neuronal ECM expression after injury or in Alzheimer’s disease has proven to be beneficial by promoting axon growth and increasing synaptic plasticity. Yet, severe impairment of ECM function can also lead to neurodegeneration. Thus, it appears that to ensure healthy neuronal function a delicate balance of ECM components must be maintained. In this paper we review the structure of PNNs and their components, such as hyaluronan, proteoglycans, core proteins, chondroitin sulphate proteoglycans, tenascins, and Hapln proteins. We also characterize the role of ECM in the functioning of the blood-brain barrier, neuronal communication, as well as the participation of PNNs in synaptic plasticity and some clinical aspects of perineuronal net impairment. Furthermore, we discuss the participation of PNNs in brain signaling. Understanding the molecular foundations of the ways that PNNs participate in brain signaling and synaptic plasticity, as well as how they change in
physiological and pathological conditions, may help in the development of new therapies for many degenerative and inflammatory diseases of the brain.

Keywords:perineuronalnets (PNNs); extracellularmatrix(ECM); synaptogenesis; neuronal communication

Directed glial differentiation and transdifferentiation for neural tissue regeneration.

Exp Neurol. 2019 Sep;319:112813. doi: 10.1016/j.expneurol.2018.08.010. Epub 2018 Aug 30.

Janowska J1, Gargas J1, Ziemka-Nalecz M1, Zalewska T1, Buzanska L2, Sypecka J3.

Author information

1 Mossakowski Medical Research Centre, Polish Academy of Sciences, NeuroRepair Department, 5, Pawinskiego str., 02-106 Warsaw, Poland.

2 Mossakowski Medical Research Centre, Polish Academy of Sciences, Stem Cell Bioengineering Unit, 5, Pawinskiego str., 02-106 Warsaw, Poland.

3 Mossakowski Medical Research Centre, Polish Academy of Sciences, NeuroRepair Department, 5, Pawinskiego str., 02-106 Warsaw, Poland. Electronic address: This email address is being protected from spambots. You need JavaScript enabled to view it..

Abstract

Glial cells which are indispensable for the central nervous system development and functioning, are proven to be vulnerable to a harmful influence of pathological cues and tissue misbalance. However, they are also highly sensitive to both in vitro and in vivo modulation of their commitment, differentiation, activity and even the fate-switch by different types of bioactive molecules. Since glial cells (comprising macroglia and microglia) are an abundant and heterogeneous population of neural cells, which are almost uniformly distributed in the brain and the spinal cord parenchyma, they all create a natural endogenous reservoir of cells for potential neurogenerative processes required to be initiated in response to pathophysiological cues present in the local tissue microenvironment. The past decade of intensive investigation on a spontaneous and enforced conversion of glial fate into either alternative glial (for instance from oligodendrocytes to astrocytes) or neuronal phenotypes, has considerably extended our appreciation of glial involvement in restoring the nervous tissue cytoarchitecture and its proper functions. The most effective modulators of reprogramming processes have been identified and tested in a series of pre-clinical experiments. A list of bioactive compounds which are potent in guiding in vivo cell fate conversion and driving cell differentiation includes a selection of transcription factors, microRNAs, small molecules, exosomes, morphogens and trophic factors, which are helpful in boosting the enforced neuro-or gliogenesis and promoting the subsequent cell maturation into desired phenotypes. Herein, an issue of their utility for a directed glial differentiation and transdifferentiation is discussed in the context of elaborating future therapeutic options aimed at restoring the diseased nervous tissue.

KEYWORDS:

Cell fate conversion; Glial cell reprogramming; Neural development transdifferentiation; Neurorepair; Pre-clinical studies; Therapeutic strategies; Tissue restoration

 

The collagen scaffold supports hiPSC-derived NSC growth and restricts hiPSC.

Front Biosci (Schol Ed). 2019 Mar 1;11:105-121.

Zychowicz M1, Pietrucha K2, Podobinska M1, Kowalska-Wlodarczyk M3, Lenart J4, Augustyniak J1, Buzanska L5.

Author information

1 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5 St, 02-106, Warsaw, Poland.

2 Department of Material and Commodity Sciences and Textile Metrology, Lodz University of Technology, Zeromskiego 116 St, 90-924, Lodz, Poland.

3 Oil and Gas Institute, National Research Institute, 25 A Lubicz St, 31-503 Cracow, Poland.

4 Department of Neurochemistry, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5 St, 02-106, Warsaw, Poland.

5 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawinskiego 5 St, 02-106, Warsaw, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it..

 

Abstract

The human induced pluripotent stem cells (hiPSC) are one of the promising candidates as patient specific cell source for autologous transplantation or modeling of diseases. The collagen (Col) scaffolds have been shown suitable to create in vitro biomimetic microenvironment for human neural stem cells, but their ability to accommodate stem cells at different stages of neural differentiation has not been verified yet. In this paper we compare lineage related hiPSC during neural differentiation for their ability to colonize Col scaffold. We have also focused on modification of collagen physicochemical properties with improved mechanical and thermal stability, without loss of its biological activity. The hiPSC expressing markers of pluripotency (OCT4, SOX2, NANOG) after neural commitment are NESTIN, GFAP, PDGFR alpha, beta- TUBULIN III, MAP-2, DCX, GalC positive. We have shown, that Col scaffold was not preferable for hiPSC culture, while the neurally committed population after seeding on Col scaffolds revealed good adhesion, viability, proliferation, along with sustaining markers of neuronal and glial differentiation. The Col scaffold-based 3D culture of hiPSC-NSCs may serve as a research tool for further translational studies.

 

Bezafibrate Upregulates Mitochondrial Biogenesis and Influence Neural Differentiation of Human-Induced Pluripotent Stem Cells.

Mol Neurobiol. 2019 Jun;56(6):4346-4363. doi: 10.1007/s12035-018-1368-2. Epub 2018 Oct 13.

Augustyniak J1, Lenart J2, Gaj P3, Kolanowska M4, Jazdzewski K3,4, Stepien PP5,6,7, Buzanska L8.

Author information

1 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.

2 Department of Neurochemistry, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.

3 Laboratory of Human Cancer Genetics, Centre of New Technologies, University of Warsaw, Warsaw, Poland.

4 Genomic Medicine, Medical University of Warsaw, Warsaw, Poland.

5 Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

6 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

7 Centre of New Technologies, University of Warsaw, Warsaw, Poland.

8 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

Abstract

Bezafibrate (BZ) regulates mitochondrial biogenesis by activation of PPAR's receptors and enhancing the level of PGC-1α coactivator. In this report, we investigated the effect of BZ on the expression of genes (1) that are linked to different pathways involved in mitochondrial biogenesis, e.g., regulated by PPAR's receptors or PGC-1α coactivator, and (2) involved in neuronal or astroglial fate, during neural differentiation of hiPSC. The tested cell populations included hiPSC-derived neural stem cells (NSC), early neural progenitors (eNP), and neural progenitors (NP). RNA-seq analysis showed the expression of PPARA, PPARD receptors and excluded PPARG in all tested populations. The expression of PPARGC1A encoding PGC-1α was dependent on the stage of differentiation: NSC, eNP, and NP differed significantly as compared to hiPSC. In addition, BZ-evoked upregulation of PPARGC1A, GFAP, S100B, and DCX genes coexist with downregulation of MAP2 gene only at the eNP stage of differentiation. In the second task, we investigated the cell sensitivity and mitochondrial biogenesis upon BZ treatment. BZ influenced the cell viability, ROS level, mitochondrial membrane potential, and total cell number in concentration- and stage of differentiation-dependent manner. Induction of mitochondrial biogenesis evoked by BZ determined by the changes in the level of SDHA and COX-1 protein, and mtDNA copy number, as well as the expression of NRF1, PPARGC1A, and TFAM genes, was detected only at NP stage for all tested markers. Thus, developmental stage-specific sensitivity to BZ of neurally differentiating hiPSC can be linked to mitochondrial biogenesis, while fate commitment decisions to PGC-1α (encoded by PPARGC1A) pathway.

KEYWORDS:

Bezafibrate; Mitochondrial biogenesis; NSC; PGC-1α; PPAR’s; hiPSC

 

Species-specific models in toxicology: in vitro epithelial barriers.

Environ Toxicol Pharmacol. 2019 Aug;70:103203. doi: 10.1016/j.etap.2019.103203. Epub 2019 Jun 1.
Bertero A1, Augustyniak J2, Buzanska L2, Caloni F3.
Author information
1 Università degli Studi di Milano, Department of Veterinary Medicine (DIMEVET) Milan, Italy.
2 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.
3 Università degli Studi di Milano, Department of Veterinary Medicine (DIMEVET) Milan, Italy. Electronic address: This email address is being protected from spambots. You need JavaScript enabled to view it..
Abstract
Species-specific in vitro epithelial barriers represent interesting predictive tools for risk assessment evaluation in toxicological studies. Moreover, these models could be applied either as stand-alone methods for the study of absorption, bioavailability, excretion, transport, effects of xenobiotics, or through an Integrated Testing Strategy. The aim of this review is to give a comprehensive overview of in vitro species-specific epithelial barrier models from bovine, dog and swine. Bovine mammary epithelial barrier as a fundamental instrument for the evaluation of the toxicant excretion, the blood brain barrier as a useful first approach in toxicological and pharmacological studies, the porcine intestinal barrier, the canine skin barrier, and finally the pulmonary barrier from bovine and swine species are described in this review.
Copyright © 2019 Elsevier B.V. All rights reserved.
KEYWORDS:
Barriers; Species-specific; Toxicology; in vitro

Species-specific models in toxicology: in vitro epithelial barriers.

Environ Toxicol Pharmacol. 2019 Aug;70:103203. doi: 10.1016/j.etap.2019.103203. Epub 2019 Jun 1.

Bertero A1, Augustyniak J2, Buzanska L2, Caloni F3.

Author information

1        Università degli Studi di Milano, Department of Veterinary Medicine (DIMEVET) Milan, Italy.

2        Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.

3        Università degli Studi di Milano, Department of Veterinary Medicine (DIMEVET) Milan, Italy. Electronic address: This email address is being protected from spambots. You need JavaScript enabled to view it..

Abstract

Species-specific in vitro epithelial barriers represent interesting predictive tools for risk assessment evaluation in toxicological studies. Moreover, these models could be applied either as stand-alone methods for the study of absorption, bioavailability, excretion, transport, effects of xenobiotics, or through an Integrated Testing Strategy. The aim of this review is to give a comprehensive overview of in vitro species-specific epithelial barrier models from bovine, dog and swine. Bovine mammary epithelial barrier as a fundamental instrument for the evaluation of the toxicant excretion, the blood brain barrier as a useful first approach in toxicological and pharmacological studies, the porcine intestinal barrier, the canine skin barrier, and finally the pulmonary barrier from bovine and swine species are described in this review.

KEYWORDS:

Barriers; Species-specific; Toxicology; in vitro

 

Organoids are promising tools for species-specific in vitro toxicological studies.

J Appl Toxicol. 2019 Jun 5. doi: 10.1002/jat.3815. [Epub ahead of print]

Augustyniak J1, Bertero A2, Coccini T3, Baderna D4, Buzanska L1, Caloni F2

Author information

1 Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.

2 Department of Veterinary Medicine (DIMEVET), Università degli Studi di Milano, Milan, Italy.

3 Laboratory of Clinical and Experimental Toxicology, Toxicology Unit, ICS Maugeri SpA-SB, IRCCS Pavia, Pavia, Italy.

4 Laboratory of Environmental Chemistry and Toxicology, Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Italy.

Abstract

Organoids are three-dimensional self-aggregating structures generated from stem cells (SCs) or progenitor cells in a process that recapitulates molecular and cellular stages of early organ development. The differentiation process leads to the appearance of specialized mature cells and is connected with changes in the organoid internal structure rearrangement and self-organization. The formation of organ-specific structures in vitro with highly ordered architecture is also strongly influenced by the extracellular matrix. These features make organoids as a powerful model for in vitro toxicology. Nowadays this technology is developing very quickly. In this review we present, from a toxicological and species-specific point of view, the state of the art of organoid generation from adult SCs and pluripotent SCs: embryonic SCs or induced pluripotent SCs. The current culture organoid techniques are discussed for their main advantages, disadvantages and limitations. In the second part of the review, we concentrated on the characterization of species-specific organoids generated from tissue-specific SCs of different sources: mammary (bovine), epidermis (canine), intestinal (porcine, bovine, canine, chicken) and liver (feline, canine).

KEYWORDS:

3D models; ESCs; iPSCs; organoids; spheroids; stem cells; toxicology

 

Reference Gene Validation via RT-qPCR for Human iPSC-Derived Neural Stem Cells and Neural Progenitors.

Mol Neurobiol. 2019 Mar 29. doi: 10.1007/s12035-019-1538-x. [Epub ahead of print]

Augustyniak J1, Lenart J2, Lipka G1, Stepien PP3, Buzanska L4.

Author information

1 Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland.

2 Department of Neurochemistry, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

3 Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, 5a Pawinskiego Str., 02-106, Warsaw, Poland.

4 Department of Stem Cell Bioengineering, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., 02-106, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

 

Abstract

Correct selection of the reference gene(s) is the most important step in gene expression analysis. The aims of this study were to identify and evaluate the panel of possible reference genes in neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP) obtained from human-induced pluripotent stem cells (hiPSC). The stability of expression of genes commonly used as the reference in cells during neural differentiation is variable and does not meet the criteria for reference genes. In the present work, we evaluated the stability of expression of 16 candidate reference genes using the four most popular algorithms: the ΔCt method, BestKeeper, geNorm and NormFinder. All data were analysed using the online tool RefFinder to obtain a comprehensive ranking. Our results indicate that NormFinder is the best tool for reference gene selection in early stages of hiPSC neural differentiation. None of the 16 tested genes is suitable as reference gene for all three stages of development. We recommend using different genes (panel of genes) to normalise RT-qPCR data for each of the neural differentiation stages.

KEYWORDS:

Gene expression reference panel; Neural Progenitor; Neural Stem Cells; Relative gene expression; human induced Pluripotent Stem Cell (hiPSC); quantitative real-time Polymerase Chain Reaction (RT-qPCR)

 

A Distinct Advantage to Intraarterial Delivery of 89Zr-Bevacizumab in PET Imaging of Mice With and Without Osmotic Opening of the Blood-Brain Barrier.

A Distinct Advantage to Intraarterial Delivery of 89Zr-Bevacizumab in PET Imaging of Mice With and Without Osmotic Opening of the Blood-Brain Barrier.

J Nucl Med. 2019 May;60(5):617-622. doi: 10.2967/jnumed.118.218792. Epub 2018 Oct 12.

Lesniak WG1, Chu C1,2, Jablonska A1,2, Du Y1, Pomper MG1, Walczak P1,2,3, Janowski M4,2,5.

Author information

1        Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins    University School of Medicine, Baltimore, Maryland.

2        Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland.

3        Department of Neurosurgery, University of Warmia and Mazury, Olsztyn, Poland; and.

4        Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland This email address is being protected from spambots. You need JavaScript enabled to view it..

5        NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

Abstract

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.

© 2019 by the Society of Nuclear Medicine and Molecular Imaging.

KEYWORDS:

brain; drug delivery; endovascular; molecular imaging; nuclear medicine

 

Biodistribution of Glial Progenitors in a Three Dimensional-Printed Model of the Piglet Cerebral Ventricular System.

Biodistribution of Glial Progenitors in a Three Dimensional-Printed Model of the Piglet Cerebral Ventricular System.

Stem Cells Dev. 2019 Apr 15;28(8):515-527. doi: 10.1089/scd.2018.0172. Epub 2019 Mar 28.

Srivastava RK1,2, Jablonska A1,2, Chu C1,2, Gregg L3, Bulte JWM1,2, Koehler RC4, Walczak P1,2,5, Janowski M1,2,6.

Author information

1        Division of MR Research, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

2        Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

3        Visualization Core Laboratory, Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

4        Department of Anesthesiology and Critical Care Medicine, Translational Tissue Engineering Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

5        Department of Neurology and Neurosurgery, University of Warmia and Mazury, Olsztyn, Poland.

6        NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

Abstract

White matter damage persists in hypoxic-ischemic newborns even when treated with hypothermia. We have previously shown that intraventricular delivery of human glial progenitors (GPs) at the neonatal stage is capable of replacing abnormal host glia and rescuing the lifespan of dysmyelinated mice. However, such transplantation in the human brain poses significant challenges as related to high-volume ventricles and long cell migration distances. These challenges can only be studied in large animal model systems. In this study, we developed a three dimensional (3D)-printed model of the ventricular system sized to a newborn pig to investigate the parameters that can maximize a global biodistribution of injected GPs within the ventricular system, while minimizing outflow to the subarachnoid space. Bioluminescent imaging and magnetic resonance imaging were used to image the biodistribution of luciferase-transduced GPs in simple fluid containers and a custom-designed, 3D-printed model of the piglet ventricular system. Seven independent variables were investigated. The results demonstrated that a low volume (0.1 mL) of cell suspension is essential to keep cells within the ventricular system. If higher volumes (1 mL) are needed, a very slow infusion speed (0.01 mL/min) is necessary. Real-time magnetic resonance imaging demonstrated that superparamagnetic iron oxide (SPIO) labeling significantly alters the rheological properties of the GP suspension, such that, even at high speeds and high volumes, the outflow to the subarachnoid space is reduced. Several other factors, including GP species (human vs. mouse), type of catheter tip (end hole vs. side hole), catheter length (0.3 vs. 7.62 m), and cell concentration, had less effect on the overall distribution of GPs. We conclude that the use of a 3D-printed phantom model represents a robust, reproducible, and cost-saving alternative to in vivo large animal studies for determining optimal injection parameters.

KEYWORDS:

CSF; MRI; bioluminescence; brain; glial progenitors; iron oxide; ventricle