Intra-arterial Administration of Human Umbilical Cord Blood Derived Cells Inversed Learning Asymmetry Resulting From Focal Brain Injury in Rat.

Background: Focal brain injury is a leading cause of serious disability significantly worsening patients' quality of life. Such damage disrupts the existing circuits, leads to motor, and cognitive impairments as well as results in a functional asymmetry. To date, there is still no therapy to effectively restore the lost functions. We examined the effectiveness of human umbilical cord blood (HUCB)-derived cells after their intra-arterial infusion following focal stroke-like brain damage. Methods: The model of stroke was performed using ouabain stereotactic injection into the right dorsolateral striatum in rats. Two days following the brain injury 107 cells were infused into the right carotid artery. The experimental animals were placed into enriched environment housing conditions to enhance the recovery process. Behavioral testing was performed using a battery of tasks visualizing motor as well as cognitive deficits for 30 days following brain injury. We assessed animal asymmetry while they were moving forward at time of testing in different tasks. Results: We found that intra-arterial infusion of HUCB-derived cells inversed lateralized performance resulting from the focal brain injury at the early stage of T-maze habit learning task training. The inversion was independent from the level of neural commitment of infused cells. The learning asymmetry inversion was observed only under specific circumstances created by the applied task design. We did not found such inversion in walking beam task, vibrissae elicited forelimb placing, the first exploration of open field, T-maze switching task as well as apomorphine induced rotations. Both the asymmetry induced by the focal brain injury and its inversion resulting from cell infusion decreased along the training. The inversion of learning asymmetry was also independent on the range of the brain damage. Conclusions: Intra-arterial infusion of HUCB-derived cells inversed lateralized performance of learning task resulting from focal brain damage. The inversion was not visible in any other of the used motor as well as cognitive tests. The observed behavioral effect of cell infusion was also not related to the range of the brain damage. Our findings contribute to describing the effects of systemic treatment with the HUCB-derived cells on functional recovery following focal brain injury.

Neurotransplantation in mice: the concorde-like position ensures minimal cell leakage and widespread distribution of cells transplanted into the cisterna magna.

The access of transplanted cells to large areas of the CNS is of critical value for cell therapy of chronic diseases associated with widespread neurodegeneration. Intrathecal cell application can match this requirement. Here we describe an efficient method for cell injection into the cisterna magna and the assessment of the cell distribution within subarachnoidal space in mice. In order to maximize cell distribution we applied a "concord-like" position, where the cisterna magna is nearly the highest point of the animal's body. A drop of saline on the needle insertion site avoided the outflow of transplanted cells from subarachnoidal space with CSF during surgery. Twenty-four hours later the preparation of the CNS with an intact dura mater by a suitable dissection technique (described in detail) revealed approx. 80% of the injected cells (100,000 cells per animal) within the subarachnoidal space ranging from the skull base (olfactory nerve to premedullary cisterns) to the IV ventricle, and to both the ventral and dorsal surfaces of the spinal cord. Thus the "concorde-like" position proved to be very useful for intrathecal cell application leading to a widespread cell distribution within the subarachnoidal space.

Evaluation of regenerative processes in the pig model of intervertebral disc degeneration after transplantation of bone marrow-derived mesenchymal stem cells.

INTRODUCTION: The pathophysiology of degenerative disc disease (DDD) is complex and not fully understood. While surgical treatment and appropriate rehabilitation offer relief of acute symptoms, there is a need to find tissue engineering strategies for intervertebral disc repair to restore healthy higher and histological structure. The purpose of this study was to estimate the survival rate of transplanted cells and their post-delivery integration level at the damage site. MATERIAL AND METHODS: We used an in vivo porcine model to investigate autogenic bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation for intervertebral disc repair. In our experiment we used a large animal model of DDD induced by percutaneous laser light deliveries. The percutaneous approach has also been used for delivery of BM-MSCs into the intervertebral disc space. RESULTS: After MSC transplantation, we observed a deceleration of the degenerative process in the intervertebral disc, relative to degenerative discs without MSC transplantation. CONCLUSIONS: By using a large animal model that mimicked the development of intervertebral degenerative disc disease, the present results are indicative of the clinical feasibility of this procedure.

Migration of human mesenchymal stem cells stimulated with pulsed electric field and the dynamics of the cell surface glycosylation.

BACKGROUND: The analysis of the stem cells' glycome dynamics at different stages of differentiation and migration makes possible the exploration of the cell surface glycans as markers of the stem cell functional status, and, in the future, compatibility between transplanted cell and host environment. OBJECTIVES: The objective of our study was to develop novel techniques of investigating cell motility and to assess whether the electric field of the therapeutic spinal cord stimulation system used in vivo contributes to the migration of human mesenchymal stem cells (hMSCs) in vitro. MATERIAL AND METHODS: We have investigated the electrotaxis of bone marrow-derived MSCs using pulsed electric field (PEF) in the range of 16-80 mV/mm and the frequency of 130 Hz and 240 Hz. The PEF-related dynamics of the cell surface glycosylation was evaluated using 6 plant lectins recognizing individual glycans. RESULTS: Pulsed electric field at physiological levels (10 mV/mm; 130 Hz) did not influence cellular motility in vitro, which may correspond to the maintenance of the transplanted cells at the lesion site in vivo. An increase of the PEF intensity and the frequency exceeding physiological levels resulted in an increase in the cellular migration rate in vitro. Pulsed electric field elevated above physiological intensity and frequency (40-80 mV/mm; 240 Hz), but not at physiological levels, resulted in changes of the cell surface glycosylation. CONCLUSIONS: We found the described approach convenient for investigations and for the in vitro modeling of the cellular systems intended for the regenerative cell transplantations in vivo. Probing cell surface glycomes may provide valuable biomarkers to assess the competence of transplanted cells.

Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro.

OBJECTIVE: The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCsCD34-) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCsCD34- results from direct conversion of hematopoietic precursors into neural-like phenotypes due to expression of overlapping genetic program or, alternatively, these neural phenotypes arise from sequential differentiation of more primitive progenitors (embryonic-like cells) preexisting in HUCB-MNCsCD34- fraction. MATERIALS AND METHODS: HUCB-MNCs negatively selected for CD34 antigens were cultured in vitro up to 14 days. Changes in stem/neural cell genes and proteins were successively evaluated during this period and after evoked neuronal differentiation of cells in the presence of RA or BDNF or cocultured with neonatal rat brain astrocytes. RESULTS: Freshly isolated HUCB-MNCsCD34- expressed pluripotent cell markers: Oct3/4, Sox2, and Rex1 genes. During 24 hours of culture the frequency of Oct3/4 immunopositive cells increased markedly with parallel enlargement of "side population" and CD133+ cell appearance. Concomitantly, cultured cells start to form aggregates and express pro-neural genes, i.e., enhanced Sox2, OTX1, Nestin, GFAP, and NF-200. During the next days of culture immunoreactions for beta-tubulin III, MAP2, GFAP, S100beta, Doublecortin, and GalC were induced with reciprocal lowering of stem cell gene and protein markers. At this stage cells successively adhered to the bottom, dispersed, and decreased proliferation rate (Ki67 expression). Additional treatments with neuromorphogenes or coculturing with rat brain primary culture induced further differentiation of these neural precursors toward more advanced neuronal phenotypes. CONCLUSIONS: HUCB-MNCs(CD34-) fraction contains embryonic-like stem/progenitor cells which increase rapidly but transiently in culture, then differentiate spontaneously after cell aggregate adhesion toward neural lineage. Neurally promoted cells from 10-14 DIV culture acquire three main neural-like phenotypes, i.e., neurons, astrocytes, and oligodendrocytes. In this respect they are promising candidates for experimental treatment of neuronal injury; however, the final proof for conversion of HUCB cells to neural cells can be obtained through transplantation experiments.

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. The neuroprotective properties of neural stem cells (NSCs) and the paracrine signaling of mesenchymal stem cells (MSCs) have been examined in multiple pre-clinical trials of ALS with promising results. The data from these initial trials indicate a reduction in the rate of disease progression. The mechanism through which stem cells achieve this reduction is of major interest. Here, we review the to-date pre-clinical and clinical therapeutic approaches employing stem cells, and discuss the most promising ones.

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.

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.

Neural commitment of cord blood stem cells (HUCB-NSC/NP): therapeutic perspectives.

Human umbilical cord blood (HUCB) is considered a promising source of neural progenitors capable of being used for cellular therapies in neurological disorders. Here we review briefly our work on the elucidation of mechanisms and development of practical standards as regards the selection, maintenance and use of cord blood derivatives for such purposes. Our results join those of other recent studies in suggesting strongly that, the generation of neural-like cells from tissue belonging to a different germ layer (such as a cord blood is) is most probably explained by reference to a discrete subpopulation of embryonic-like stem cells of pluripotent characteristics. Such cells identified in cord blood through their expression of specific genetic and protein markers can be expanded in vitro and directed toward neurally-committed progenitors differentiating further into more mature neuron-like or macroglia-like cell phenotypes. From this HUCB-derived neural progenitor fraction a novel neural-like stem cell line (HUCB-NSC) has been developed, and characterized in respect of in vitro and in vivo (post-transplantation) properties.

Epigenetic and molecular signature of human umbilical cord blood-derived neural stem cell (HUCB-NSC) neuronal differentiation.

In the context of cell therapy, the epigenetic status of core stemness transcription factor (STF) genes regulating the cell proliferation/differentiation program is of primary interest. Our results confirmed that in vitro differentiation of the umbilicalcord-blood-derived-neural-stem-cells (HUCB-NSC) coincides with the progressive down-regulation of Oct3/4 and Nanog gene expression. Consistently and in parallel with the repression of gene transcription, a substantial increase in the mosaic cytosine methylation CpG dinucleotide was observed in the promoter regions of these STF genes. However none of the histone-H3 post-translational-modifications (PTM) known to be associated with transcriptionally active genes (H3Ac and H3K4me3) or repressed genes (H3K9me3 and H3K27me3) seemed to vary in relation to the progression of cell differentiation and down-regulation of STF genes. This indicates an uncoupling between STF gene expression and above mentioned histone PTMs. In contrast, the overall methylation of nuclear chromatin at repressive histone H3K9me3 was significantly higher than H3K4 trimetylation in expanding HUCB-NSC cultures and then increases through the progression of cell differentiation. These observations suggest different epigenetic programs of gene repression realized in the cell nuclei of differentiating HUCB-NSC cultures with uneven involvement of the repressive histone PTMs.

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 10(6) 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.

Aggregation-promoted expansion of neuraly committed human umbilical cord blood progenitors in vitro.

Human umbilical cord blood (HUCB) has been established as a promising source of hematopoietic as well as various non- hematopoietic stem cell populations. This offers numerous advantages of HUCB stem/progenitor cells for therapies, however in vitro conditions that contribute to long term propagation of proliferating undifferentiated cells have not yet been established. Here we evaluate culture conditions promoting spheroid aggregates/neurospheres formation which, together with serum withdrawal and mitogenes treatments in strictly defined media, maintain population of HUCB progenitor cells in undifferentiated and dividing state exhibiting neurogenic potential in vitro. Our results indicate that formation and maintenance of three-dimensional aggregates enhanced by cell culture rotating motion, is crucial for high and prolonged expression of genes and proteins characteristic for cord blood stem cells and their further neural commitment.

Systemic treatment of focal brain injury in the rat by human umbilical cord blood cells being at different level of neural commitment.

The aim of the study was to evaluate therapeutic effectiveness of intra-arterial infusion of human umbilical cord blood (HUCB) derived cells at different stages of their neural conversion. Freshly isolated mononuclear cells (D-0), neurally directed progenitors (D-3) and neural-like stem cells derived from umbilical cord blood (NSC) were compared. Focal brain damage was induced in rats by stereotactic injection of ouabain into dorsolateral striatum Three days later 10(7) of different subsets of HUCB cells were infused into the right internal carotid artery. Following surgery rats were housed in enriched environment for 30 days. Behavioral assessment consisted of tests for sensorimotor deficits (walking beam, rotarod, vibrissae elicited forelimb placing, apomorphine induced rotations), cognitive impairments (habit learning and object recognition) and exploratory behavior (open field). Thirty days after surgery the lesion volume was measured and the presence of donor cells was detected in the brain at mRNA level. At the same time immunohistochemical analysis of brain tissue was performed to estimate the local tissue response of ouabain injured rats and its modulation after HUCB cells systemic treatment. Functional effects of different subsets of cord blood cells shared substantial diversity in various behavioral tests. An additional analysis showed that D-0 HUCB cells were the most effective in functional restoration and reduction of brain lesion volume. None of transplanted cord blood derived cell fractions were detected in rat's brains at 30(th) day after treatment. This may suggest that the mechanism(s) underlying positive effects of HUCB derived cell may concern the other than direct neural cell supplementation. In addition increased immunoreactivity of markers indicating local cells proliferation and migration suggests stimulation of endogenous reparative processes by HUCB D-0 cell interarterial infusion.

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.

Generation of functional neural artificial tissue from human umbilical cord blood stem cells.

Stem cell-based regenerative neurology is an emerging concept for treatment of diseases of central nervous system. Among variety of proposed procedures, one of the most promising is refilling of cystic cavities of injured brain parenchyma with artificial neural tissue. Recent studies revealed that after allogenic transplantation in rodents these tissue-engineered entities were shown efficient in repair of hypoxic/ischemic brain injury. Human umbilical cord blood (HUCB) was recognized to be an efficient and noncontroversial source of neural stem cells (NSC). The main purpose of this study was to generate HUCB-derived neural artificial tissue and investigate their functional properties. Neural organoids formed on human-originated biodegradable scaffolds within 3 weeks and resembled niche structure where immature stem cells (Oct4+ and Sox2+) and proliferating neuroblasts (Nestin+, GFAP+, and Ki67+) were present. Such aggregates were placed on multi-electrode chips and differentiated toward mature neurons (TUJ1+ and MAP2+). These three-dimensional aggregates in contrast to two-dimensional cultures formed functional circuits and generated spontaneous field/action potentials. Our results indicate that three-dimensional environment facilitates maturation of HUCB-derived NSC what should be considered regarding regenerative medicine application.