Krystyna Domanska-Janik

Voltage‐Sensitive and Ligand‐Gated Channels in Differentiating Neural Stem–Like Cells Derived from the Nonhematopoietic Fraction of Human Umbilical Cord Blood

Fetal cells with the characteristics of neural stem cells (NSCs) can be derived from the nonhematopoietic fraction of human umbilical cord blood (HUCB), expanded as a nonimmortalized cell line (HUCB‐NSC), and further differentiated into neuron‐like cells (HUCB‐NSCD); however, the functional and neuronal properties of these cells are poorly understood. To address this issue, we used whole‐cell patch‐clamp recordings, gene microarrays, and immunocytochemistry to identify voltage‐gated channels and ligand‐gated receptors on HUCB‐NSCs and HUCB‐NSCDs. Gene microarray analysis identified genes for voltage‐dependent potassium and sodium channels and the neurotransmitter receptors acetylcholine (ACh), γ‐aminobutyric acid (GABA), glutamate, glycine, 5‐hydroxytryptamine (5‐HT), and dopamine (DA). Several of these genes (GABA‐A, glycine and glutamate receptors, voltage‐gated potassium channels, and voltage‐gated sodium type XII alpha channels) were not expressed in the HUCB mono‐nuclear fraction (HUCB‐MC), which served as a starting cell population for HUCB‐NSC. HUCB‐NSCD acquired neuronal phenotypes and displayed an inward rectifying potassium current (Kir) and an outward rectifying potassium current (IK+). Kir was present on most HUCB‐NSCs and HUCB‐NSCDs, whereas IK+ was present only on HUCB‐NSCDs. Many HUCB‐NSCDs were immunopositive for glutamate, glycine, nicotinic ACh, DA, 5‐HT, and GABA receptors. Kainic acid (KA), a non–N‐methyl‐D‐asparate (NMDA) glutamate‐receptor agonist, induced an inward current in some HUCB‐NSCDs. KA, glycine, DA, ACh, GABA, and 5‐HT partially blocked Kir through their respective receptors. These results suggest that HUCB‐NSCs differentiate toward neuron‐like cells, with functional voltage‐ and ligand‐gated channels identified in other neuronal systems.

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

Effect of brain ischemia on protein kinase C.

Abstract: We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6‐min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13‐dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane‐bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane‐bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca2+‐dependent, antiprotease‐insensitive down‐regulation of PKC during incubation. This down‐regulation was significantly enhanced by prior phorbol (PDBu) treatment. It can be postulated that in the ischemic gerbil hippocampus model as well as during phorbol stimulation of the synaptoneurosomal fraction in vitro, the common phenomenon was an initial PKC activation (translocation) followed by a subsequent enhancement of Ca2+‐dependent enzyme inhibition. Experimental data suggest that the inhibition is localized in the membrane compartment.

Neurogenic potential of human umbilical cord blood: Neural-like stem cells depend on previous long-term culture conditions

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB‐NPs). Due to further expansion of these cells we have established the first human umbilical cord blood‐derived neural‐like stem cell line (HUCB‐NSC) growing in serum‐free (SF) or low‐serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB‐NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB‐NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co‐cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co‐cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial‐specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB‐NSC on the neurogenic signals provided by surrounding environment in vitro.

Opposite reaction of ERK and JNK in ischemia vulnerable and resistant regions of hippocampus: involvement of mitochondria.

Delayed ischemic death of neurones is observed selectively in CA1 region of hippocampus at 3-4 days of reperfusion. Signals generated immediately during and after ischemia are further propagated by a variety of kinases, proteases and phosphatases. Tissue samples from dorsal (vulnerable) and abdominal (resistant) parts of gerbil hippocampi were collected to determine the activation state of key signaling molecules: Akt, Raf-1, JNK, ERK1/2 in the course of reperfusion after 5 min of global cerebral ischemia. Western blot analysis of phosphorylated forms of the kinases revealed persistent activation of JNK, being limited mostly to vulnerable CA1 region. On the contrary, activation of ERK, although observed transiently in both parts, was enhanced for a longer time in the abdominal hippocampus. The levels of the active/phosphorylated Akt and Raf-1 kinases did not change significantly during the recovery period. No significant correlation between postischemic JNK activation and c-Jun phosphorylation or its contribution to AP1-like complex formation was found. In contrast, the amount of active JNK linked with mitochondrial membranes was significantly increased and preceded neuronal death in CA1. In the same period of time the AP1 complex, augmented in CA1 region, did not appear to contain a classical c-Fos protein. These results are consistent with the theory that either long-lasting activation of JNK and/or contrasting ERK and JNK activities in critical time of reperfusion, contribute to selective apoptosis of CA1 neurons. This, in connection with the translocation of activated JNK to mitochondria and time/regional differences in AP1 binding protein complexes can affect final postischemic outcome.

Long-term MRI cell tracking after intraventricular delivery in a patient with global cerebral ischemia and prospects for magnetic navigation of stem cells within the CSF.

Background The purpose of the study was to evaluate the long-term clinical tracking of magnetically labeled stem cells after intracerebroventricular transplantation as well as to investigate in vitro feasibility for magnetic guidance of cell therapy within large fluid compartments. Method After approval by our Institutional Review Board, an 18-month-old patient, diagnosed as being in a vegetative state due to global cerebral ischemia, underwent cell transplantation to the frontal horn of the lateral ventricle, with umbilical cord blood-derived stem cells labeled with superparamagnetic iron oxide (SPIO) contrast agent. The patient was followed over 33 months with clinical examinations and MRI. To evaluate the forces governing the distribution of cells within the fluid compartment of the ventricular system in vivo, a gravity-driven sedimentation assay and a magnetic field-driven cell attraction assay were developed in vitro. Results Twenty-four hours post-transplantation, MR imaging (MRI) was able to detect hypointense cells in the occipital horn of the lateral ventricle. The signal gradually decreased over 4 months and became undetectable at 33 months. In vitro, no significant difference in cell sedimentation between SPIO-labeled and unlabeled cells was observed (p = NS). An external magnet was effective in attracting cells over distances comparable to the size of human lateral ventricles. Conclusions MR imaging of SPIO-labeled cells allows monitoring of cells within lateral ventricles. While the initial biodistribution is governed by gravity-driven sedimentation, an external magnetic field may possibly be applied to further direct the distribution of labeled cells within large fluid compartments such as the ventricular system.

Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner

BACKGROUND AIMS As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly-derived MSCs (WJ-MSCs). METHODS Primary cultures growing in 21% oxygen were either transferred into 5% O2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α-3α were evaluated. RESULTS Lowering O2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. CONCLUSIONS A physiologically relevant microenvironment of 5% O2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α-mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

Neuronal Differentiation of Human Umbilical Cord Blood Neural Stem-Like Cell Line

The expanding population of neural stem/progenitor cells can be selected from human cord blood nonhematopoietic (CD34-negative) mononuclear fraction. Due to repeated expansion and selection of these cells we have established the first clonogenic, nonimmortalized human umbilical cord blood neural stem-like cell (HUCB-NSC) line. This line can be maintained at different stages of neural progenitor development by the presence of trophic factors, mitogens and neuromorphogens in culture media. Neurogenic potential of HUCB-NSC was established for serum-free and low-serum cultured cells. Commitment of HUCB-NSC by serum was shown to be important for the optimal response to the signals provided by surrounding environment in vitro. Enhanced neuronal differentiation induced by dBcAMP treatment was accompanied by expression of several functional proteins including glutamatergic, GABAergic, dopamine, serotonin and acetylcholine receptors, which was shown by microarray, immunocytochemistry and electrophysiology. Electrophysiological studies, whole-cell patch-clamp recordings, revealed in differentiated HUCB-NSC two types of voltage-sensitive and several ligand-gated currents typical for neuronal cells. The above HUCB-NSC characteristic conceivably implicates that cord blood-derived progenitors could be effectively differentiated into functional neuron-like cells in vitro.

Transient forebrain ischemia modulates signal transduction from extracellular matrix in gerbil hippocampus.

Cell adhesion to the extracellular matrix (ECM) functions as a survival factor and disruption of cell-ECM interaction can lead to cell death. Our previous study has demonstrated ischemia-induced enhancement of activity of extracellular metalloproteinases, which might result in the alteration of adhesive contact with ECM and affect the intracellular signaling pathway. The enzyme thought to play a major role in conveying survival signals from ECM to the cell interior is focal adhesion kinase (pp125(FAK)). In the present study, the temporal relation between activation of extracellular metalloproteinases (MMP-2 and MMP-9), degradation of extracellular matrix protein laminin and the expression of pp125(FAK) after 5 min of global ischemia in gerbil hippocampus were investigated. While significant activation of both investigated metalloproteinases occurred in the course of reperfusion, only changes in MMP-9 activity were correlated with degradation of laminin. These ischemia-induced extracellular events coincide temporarily with proteolytic modification of FAK protein and diminished level of its phosphorylated form, to about 50% of the initial value. These results are indicative of an involvement of ECM-pp125(FAK) signaling pathway in ischemia-induced neuronal degeneration.

Intracerebroventricular Transplantation of Cord Blood-Derived Neural Progenitors in a Child with Severe Global Brain Ischemic Injury:

Transplantation of neural stem/precursor cells has recently been proposed as a promising, albeit still controversial, approach to brain repair. Human umbilical cord blood could be a source of such therapeutic cells, proven beneficial in several preclinical models of stroke. Intracerebroventricular infusion of neutrally committed cord blood-derived cells allows their broad distribution in the CNS, whereas additional labeling with iron oxide nanoparticles (SPIO) enables to follow the fate of engrafted cells by MRI. A 16-month-old child at 7 months after the onset of cardiac arrest-induced global hypoxic/ischemic brain injury, resulting in a permanent vegetative state, was subjected to intracerebroventricular transplantation of the autologous neutrally committed cord blood cells. These cells obtained by 10-day culture in vitro in neurogenic conditions were tagged with SPIO nanoparticles and grafted monthly by three serial injections (12 × 10(6) cells/0.5 ml) into lateral ventricle of the brain. Neural conversion of cord blood cells and superparamagnetic labeling efficiency was confirmed by gene expression, immunocytochemistry, and phantom study. MRI examination revealed the discrete hypointense areas appearing immediately after transplantation in the vicinity of lateral ventricles wall with subsequent lowering of the signal during entire period of observation. The child was followed up for 6 months after the last transplantation and his neurological status slightly but significantly improved. No clinically significant adverse events were noted. This report indicates that intracerebroventricular transplantation of autologous, neutrally committed cord blood cells is a feasible, well tolerated, and safe procedure, at least during 6 months of our observation period. Moreover, a cell-related MRI signal persisted at a wall of lateral ventricle for more than 4 months and could be monitored in transplanted brain hemisphere.