Aleksandra Habich

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.

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.

MR monitoring of minimally invasive delivery of mesenchymal stem cells into the porcine intervertebral disc.

Purpose Bone marrow stem cell therapy is a new, attractive therapeutic approach for treatment of intervertebral disc (IVD) degeneration; however, leakage and backflow of transplanted cells into the structures surrounding the disc may lead to the formation of undesirable osteophytes. The purpose of this study was to develop a technique for minimally invasive and accurate delivery of stem cells. Methods Porcine mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIO, Molday ION rhodamine) and first injected into the explanted swine lumbar IVD, followed by ex vivo 3T MRI. After having determined sufficient sensitivity, IVD degeneration was then induced in swine (n=3) by laser-evaporation. 3 x 106 SPIO-labeled cells embedded within hydrogel were injected in 2 doses using a transcutaneous cannula and an epidural anesthesia catheter. T2-weighted MR images were obtained at 3T before and immediately after cell infusion. Two weeks after injection, histological examination was performed for detection of transplanted cells. Results MSCs were efficiently labeled with Molday ION rhodamine. Cells could be readily detected in the injected vertebral tissue explants as distinct hypointensities with sufficient sensitivity. MR monitoring indicated that the MSCs were successfully delivered into the IVD in vivo, which was confirmed by iron-positive Prussian Blue staining of the tissue within the IVD. Conclusion We have developed a technique for non-invasive monitoring of minimally invasive stem delivery into the IVD at 3T. By using a large animal model mimicking the anatomy of IVD in humans, the present results indicate that this procedure may be clinically feasible.

Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system

Stem cell therapy for neurological disorders reached a pivotal point when the efficacy of several cell types was demonstrated in small animal models. Translation of stem cell therapy is contingent upon overcoming the challenge of effective cell delivery to the human brain, which has a volume ∼1000 times larger than that of the mouse. Intra-arterial injection can achieve a broad, global, but also on-demand spatially targeted biodistribution; however, its utility has been limited by unpredictable cell destination and homing as dictated by the vascular territory, as well as by safety concerns. We show here that high-speed MRI can be used to visualize the intravascular distribution of a superparamagnetic iron oxide contrast agent and can thus be used to accurately predict the distribution of intra-arterial administered stem cells. Moreover, high-speed MRI enables the real-time visualization of cell homing, providing the opportunity for immediate intervention in the case of undesired biodistribution.

Stem cells for ALS: An overview of possible therapeutic approaches

Amyotrophic lateral sclerosis (ALS) is an unusual, fatal, neurodegenerative disorder leading to the loss of motor neurons. After diagnosis, the average lifespan ranges from 3 to 5 years, and death usually results from respiratory failure. Although the pathogenesis of ALS remains unclear, multiple factors are thought to contribute to the progression of ALS, such as network interactions between genes, environmental exposure, impaired molecular pathways and many others.

Function of ID1 protein in human cord blood-derived neural stem-like cells

The effect of dominant‐negative regulator of basic helix‐loop‐helix (bHLH) transcription factors, an ID1 protein, on growth and differentiation of neural stem‐like cell line derived from human umbilical cord blood (HUCB‐NSC) was investigated. This nontransformed, mesodermal germ layer‐originated line contains high levels of ID1 protein, whose intercellular distribution reflects HUCB‐NSC differentiation status. Whereas cells remained undifferentiated and self‐renewing in serum‐free (SF) cultures, ID1 protein, although highly expressed, did not attain cell nuclei and was localized mainly in cytoplasm. In long‐term‐expanded cultures of partially committed (primed) HUCB‐NSC grown in a low serum concentration (LS cultures) ID1 protein became translocated toward cell nuclei. Further neuronal differentiation of the cells, either spontaneous in the presence of serum or induced by neuromorphogens (dBcAMP, RA), resulted in almost complete depletion of ID1 mRNA and protein. Accordingly, HUCB‐NSC transfectants overexpressing the ID1 gene were significantly inhibited in their differentiation. Notably, only neuronal and not glial development was affected after ID1 overexpression. A similar gain‐of‐function effect of ID1 transfection was observed in human NSC‐like line (DEV) of medullobastoma origin, which is constitutively devoid of ID1 expression. Thus, our results on HUCB‐NSC confirm further its neural‐specific behavior and the crucial role of ID1 protein as a potent negative regulator of neural stem cell differentiation, pointing out that this protein distribution between cytoplasmic and nuclear cell compartments can be one of the most important steps in differentiation signal transduction.

The Perspectives of Stem Cell-Based Therapy in Neurological Diseases

The impairment of function of Central Nervous System (CNS) due to the loss of nervous cells is the crucial feature of so called neurological degenerative diseases, including: Parkinson disease (PD), Alzheimer disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Huntington Disease (HD). The social importance of treatment of those two first pathologies is increasing contemporary as a result of the aging of population. Multiple Sclerosis (MS) is another devastating neurological disease in which not only myelin sheet but also neuronal degeneration occurs (Brain 132(Pt 5):1175–1189, 2009). Until now there is no effective treatment, although during last decades the diagnostic possibilities dramatically improved. It is understandable that new opportunities of the use of stem cell progenitors of neurons are the topics in the developing research. There are also perspectives for implementation of the stem cells transplantation in the treatment of loss of neurons due to the brain or spinal cord damage, as a result of the stroke and mechanical injury. In human, the early transplantation stem cells trials present a huge variety of outcomes ranging from significant clinical benefit to worsening of symptoms with severe side effects. As the pathophysiology differs in PD, ALS, MS and stroke, different cell sources for transplantation might be required for optimal clinical improvement. Elementary examination is compulsory before stem cell transplantation therapy can become a realistic clinical treatment. Recently, the overall goal for many laboratories in their research became to understand the function of human brain stem cells and how they may play a role in the origin of brain tumors. Understanding the relationship between the genesis of brain tumors and the potential interventions using stem cells are of greatest importance and has been also recently a topic for many publications.

The feasibility of the CD271+ and CD271– mesenchymal stromal cell enrichment toward nucleus pulposus-like cells

INTRODUCTION Factors promoting nerve cell ingrowth are considered responsible for chronic back pain resulting from the intervertebral disc degeneration (IDD). One of the recent exploratory IDD treatments is stem cell transplantation therapy. The CD271 (low-affinity nerve growth factor receptor) has been identified as a mark-er of the most homogeneous mesenchymal stem cell (MSC) subset. It is capable of promoting differentiation along adipogenic, osteogenic and chondrogenic lineages and producing significantly higher levels of cytokines as compared to the total population of plastic adherence-mesenchymal stem cells (PA-MSCs). We investigated the ability of CD271+ MSCs to differentiate into chondrocyte-like cells of the nucleus pulposus (NP) of intervertebral disc. We also examined CD271- MSCs, using PA-MSCs as a control cell population. MATERIAL AND METHODS Bone marrow derived PA-MSCs and its two subsets, CD271- MSCs and CD271+ MSCs, were seeded in collagen scaffolds. After two weeks of growth in NP-differentiation medium, RNA was isolated from cells-scaffold constructs and was analyzed by q-PCR for expression of NP markers. Glycosaminoglycans were analyzed biochemically directly in cells-scaffold constructs. RESULTS Expression of NP markers - extracellular matrix components such as aggrecan, collagen type II and glycosaminoglycans on both RNA and the protein levels - was significantly higher in CD271- MSCs compared to the CD271+ MSCs and PA-MSCs cell populations. CONCLUSIONS CD271- MSCs may be superior candidates for NP restorative treatment compared to CD271+ MSCs and PA-MSCs due to their ability of expressing NP-supporting extracellular matrix components at levels higher than the other two studied MSC subsets.

Methodology for Assessing the Degree of Degeneration of the Porcine Intervertebral Discs Based on Magnetic Resonance Imaging

Degeneration of intervertebral discs can lead to various health problems, among which low back pain is the most common. Studies concerning methods of dealing with that problem are being conducted and magnetic resonance imaging is one of the most popular methods of assessing their progress. Many of these studies are based on animal models. The paper links these two facts and so it contains the description of methodology that can be used for assessing the degree of degeneration of the porcine intervertebral discs based on scans obtained in MRI study. Proposed method uses Otsu method of automatic thresholding and next basic operators of morphological image processing. The outcome is binary mask giving direct information about locations of intervertebral discs in MRI scans. The obtained mask can be also used to separate locations of intervertebral discs in following MRI studies of the same animals, in cases where they are much harder to delineate due to their degeneration. The paper shows also exemplary results demonstrating usefulness of proposed methodology for the evaluation of discs treatment using mesenchymal stem cells.