Joanna Sypecka

A Human Stem Cell-Based Model for Identifying Adverse Effects of Organic and Inorganic Chemicals on the Developing Nervous System

The aim of our study was to investigate whether a human neural stem cell line derived from umbilical cord blood (HUCB‐NSC) can serve as a reliable test model for developmental neurotoxicity (DNT). We assessed the sensitivity of HUCB‐NSCs at different developmental stages to a panel of neurotoxic (sodium tellurite, methylmercury chloride, cadmium chloride, chlorpyrifos, and L‐glutamate) and non‐neurotoxic (acetaminophen, theophylline, and D‐glutamate) compounds. In addition, we investigated the effect of some compounds on key neurodevelopmental processes like cell proliferation, apoptotic cell death, and neuronal and glial differentiation. Less differentiated HUCB‐NSCs were generally more sensitive to neurotoxicants, with the notable exception of L‐glutamate, which showed a higher toxicity to later stages. The relative potencies of the compounds were: cadmium chloride > methylmercury chloride ≫ chlorpyrifos ≫ L‐glutamate. Fifty nanomolar methylmercury chloride (MeHgCl) inhibited proliferation and induced apoptosis in early‐stage cells. At the differentiated stage, 1 μM MeHgCl induced selective loss of S100β‐expressing astrocytic cells. One millimolar L‐glutamate did not influence the early stages of HUCB‐NSC development, but it affected late stages of neuronal differentiation. A valuable system for in vitro DNT assessment should be able to discriminate between neurotoxic and non‐neurotoxic compounds and show different susceptibilities to chemicals according to developmental stage and cell lineage. Although not exhaustive, this work shows that the HUCB‐NSC model fulfils these criteria and may serve as a human in vitro model for DNT priority setting. STEM CELLS 2009;27:2591–2601

The potential role of metalloproteinases in neurogenesis in the gerbil hippocampus following global forebrain ischemia.

Background Matrix metalloproteinases (MMPs) have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1) and resistant (dentate gyrus-DG) to ischemic injury areas of the brain hippocampus. Principal Findings We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs) toward the neuronal lineage. Conclusion Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus.

The Neuroprotective Effect Exerted by Oligodendroglial Progenitors on Ischemically Impaired Hippocampal Cells

Oligodendrocyte progenitor cells (OPCs) are the focus of intense research for the purpose of cell replacement therapies in acquired or inherited neurodegenerative disorders, accompanied by ongoing hypo/demyelination. Recently, it has been postulated that these glia-committed cells exhibit certain properties of neural stem cells. Advances in stem cell biology have shown that their therapeutic effect could be attributed to their ability to secret numerous active compounds which modify the local microenvironment making it more susceptible to restorative processes. To verify this hypothesis, we set up an ex vivo co-culture system of OPCs isolated from neonatal rat brain with organotypic hippocampal slices (OHC) injured by oxygen-glucose deprivation (OGD). The presence of OPCs in such co-cultures resulted in a significant neuroprotective effect manifesting itself as a decrease in cell death rate and as an extension of newly formed cells in ischemically impaired hippocampal slices. A microarray analysis of broad spectrum of trophic factors and cytokines expressed by OPCs was performed for the purpose of finding the factor(s) contributing to the observed effect. Three of them—BDNF, IL-10 and SCF—were selected for the subsequent functional assays. Our data revealed that BDNF released by OPCs is the potent factor that stimulates cell proliferation and survival in OHC subjected to OGD injury. At the same time, it was observed that IL-10 attenuates inflammatory processes by promoting the formation of the cells associated with the immunological response. Those neuroprotective qualities of oligodendroglia-biased progenitors significantly contribute to anticipating a successful cell replacement therapy.

The organotypic longitudinal spinal cord slice culture for stem cell study.

The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being cultured in vitro for up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriate in vitro model for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.

A simple, xeno-free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation.

Oligodendrocyte progenitors (OPCs) are ranked among the most likely candidates for cell‐based strategies aimed at treating neurodegenerative diseases accompanied by dys/demyelination of the central nervous system (CNS). In this regard, different sources of stem cells are being tested to elaborate xeno‐free protocols for efficient generation of OPCs for clinical applications. In the present study, neural stem cells of human umbilical cord blood (HUCB‐NSCs) have been used to derive OPCs and subsequently to differentiate them into mature, GalC‐expressing oligodendrocytes. Applied components of the extracellular matrix (ECM) and the analogues of physiological substances known to increase glial commitment of neural stem cells have been shown to significantly increase the yield of the resulting OPC fraction. The efficiency of ECM components in promoting oligodendrocyte commitment and differentiation prompted us to investigate the potential role of gelatinases in those processes. Subsequently, endogenous and ECM metalloproteinases (MMPs) activity has been compared with that detected in primary cultures of rat oligodendrocytes in vitro, as well as in rat brains in vivo. The data indicate that gelatinases are engaged in gliogenesis both in vitro and in vivo, although differently, which presumably results from distinct extracellular conditions. In conclusion, the study presents an efficient xeno‐free method of deriving oligodendrocyte from HUCB‐NSCs and analyses the engagement of MMP‐2/MMP‐9 in the processes of cell commitment and maturation. Copyright

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.

Therapeutic Strategies for Leukodystrophic Disorders Resulting from Perinatal Asphyxia: Focus on Myelinating Oligodendrocytes

Perinatal asphyxia results from the action of different risk factors like complications during pregnancy, preterm delivery, or long and difficult labor. Nowadays, it is still the leading cause of neonatal brain injury known as hypoxic-ischemic encephalopathy (HIE) and resulting neurological disorders. A temporal limitation of oxygen, glucose, and trophic factors supply results in alteration of neural cell differentiation and functioning and/or leads to their death. Among the affected cells are oligodendrocytes, responsible for myelinating the central nervous system (CNS) and formation of white matter. Therefore, one of the major consequences of the experienced HIE is leukodystrophic diseases resulting from oligodendrocyte deficiency or malfunctioning. The therapeutic strategies applied after perinatal asphyxia are aimed at reducing brain damage and promoting the endogenous neuroreparative mechanisms. In this review, we focus on the biology of oligodendrocytes and discuss present clinical treatments in the context of their efficiency in preserving white matter structure and preventing cognitive and behavioral deficits after perinatal asphyxia.

Mesenchymal cells of umbilical cord and umbilical cord blood as a source of human oligodendrocyte progenitors.

Abstract Human oligodendrocyte progenitor cells (OPCs) could be generated from stem cells by their directed differentiation and used for curing dis/demyelinating diseases and CNS injures. However the both efficient and easily accessible stem cell sources are looked for to gain biological material for further steps of OPC derivation. The mesenchymal stem cells inhabiting compartments of umbilical cord, Whartons jelly and blood in cord vessels, were shown to give rise to oligodendrocyte-biased cells after application of designed protocols. Here we present foregoing achievements in oligodendrocyte generation from stem cells of umbilical cord, either fresh or cryopreserved, and their utility for cellular therapies. The protocols are being gradually improved to clinical standards, i.e. towards procedures based on xeno-free chemical analogs of physiological substances, known to be engaged in OPC commitment and differentiation in vivo. The beneficial role of OPC engraftment is broad and often results in clinical improvement of animal models. There is some evidence that grafted OPCs contribute to remyelination in host. Taken together, OPC-based cellular therapies seem to be a promising therapeutic approach and cells of umbilical cord could serve as a cell source for oligodendrocyte derivation.

OGD induced modification of FAK- and PYK2-coupled pathways in organotypic hippocampal slice cultures.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase (PYK2) are two related non-receptor tyrosine kinases which are thought to play a role in transducing extracellular matrix (ECM)-derived survival signals into cells. The functions of FAK and PYK2 are linked to autophosphorylation of their specific tyrosine residues, Tyr-397 in FAK and Tyr-402 in PYK2, and then association with different signalling proteins which mediate activation of downstream targets such as ERK and JNK mitogen-activated kinase cascades. Thus, modulation of FAK as well as PYK2 autophosphorylation may affect several intracellular pathways and may participate in a variety of pathological settings. The present study provides a systematic investigation of the influence of experimental ischemia, induced by oxygen-glucose-deprivation, on the FAK- and PYK2-mediated signalling in organotypic hippocampal slice cultures. OGD induced primary down-regulation of FAK and PYK2 autophosphorylation (at Tyr 397 and Tyr 402, respectively) at 24-48 h of reoxygenation was accompanied by the diminution of phosphorylation/activation of Src and JNK. In contrast, the activity of Akt and ERK1/2 remained on the control level. It indicates that Akt kinase as well as ERK1/2 does not interfere with OGD-induced neuronal damage. The inhibition of the early step of FAK and PYK2 activation demonstrated by the decrease of tyrosine autophosphorylation may comprise an important portion of the response expressed by modulation of some coupled signal transduction pathways.

Oligodendrocyte development in PLP“pt” mutant rabbits: glycolipid antigens and PLP gene expression

Paralytic tremor (pt), a hereditary neurological disorder of rabbits is a recessive, X-linked point mutation of the gene for proteolipid protein (PLP) biosynthesis. This mutation results in substitution of histidine36 by glutamine in the PLP molecule and produces severe hypomyelination. In the present study, we investigated the developmental expression of myelin - oligodendrocyte - specific glycolipid markers by means of ELISA assay. While immunoreactivity with antibodies recognising proligodendroblast (POA) antigen was unchanged, only minute amounts of the other glycolipid markers characteristic for more advanced stages of OLs maturation, such as 04 and 01 antigens, were expressed inpt brain. The degree of down-regulation was similar to that for MBP. Concomitantly, the level ofin situ expression of the mutated PLP gene mRNA in glial cells of 14 day oldpt brain was found to be as high as in age-matched controls. Northern blot analysis of developmental PLP gene expression showed a significant deficit of this message inpt brain, but only at more advanced developmental stages. However, aside from changes in myelin structure, no changes in glial cell number or morphology were evident by light microscopic examination ofpt mutants. In contrast, electron microscopy revealed substantial abnormalities inpt oligodendrocyte cytoarchitecture, indicating functional impairment of intracellular transport and utilisation of myelin constituents. Thus, only POA expression is positively correlated with the unchanged content of OLs inpt brain, whereas decreases of 04 and 01 antigens, together with MBP immunoreactivity, are indicators of the degree of hypomyelination. Furthermore, oligodendrocyte differentiation appears to proceed normally inpt mutant brain up to the stage of PLP gene expression. Then, due to intracellular accumulation of this abnormal gene product, synthesis of PLP as well as the other myelin-specific constituents is inhibited by a “feed-back” control mechanism.