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1

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

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Habich A., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
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.
2

Changes in pattern of DNA methylation in DE promoter region of Oct3/4 gene before and after neural differentiation of HUCB-NSC

81%
Habich A., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
Umbilical cord blood is considered as a promising source of stem cells capable of self-renewal and differentiation into different cell types, including neural. Differentiation processes are governed by microenvironmental cues and by unique molecular mechanisms, where epigenetic changes of the chromatin play an important role. Emerging evidence suggests, that changes in expression of so called “stemness” gene, like Oct3/4, are associated with the specific epigenetic modifications of gene promoter. Methylation status of Oct3/4 and Nanog promoters correlates strongly with their ability to be expressed. The promoters are unmethylated in pluripotent stem cells, where those genes are expressed, and almost fully methylated in differentiated cells, where Oct3/4 and Nanog are silenced. The aim of the study was to analyze the DE (Distal Enhancer) promoter region’s methylation pattern in Oct3/4 gene in HUCB-NSC (Human Umbilical Cord Blood - Neural Stem Cells) line comparing to hESC (Human Embryonic Stem Cells) and also changes caused by neural differentiation of HUCB-NSC. Materials and Methods. HUCB-NSC were cultured in serum free, low serum (2% FBS) and in differentiating medium containing dBcAMP (300 µM) in the density of 5x104 cells per cm2 in standard conditions. After 14 DIV DNA from harvested cells was isolated. Methylation status of gene DE promoter region was analyzed by sodium bisulfite reaction. To analyze sequence of obtained PCR fragment subcloning into pGEM-T easy vector and sequencing was performed (at least 10 individual clones). DNA of hESC was received from Prof. Dvorak laboratory in Brno. Results. Methylation pattern of Oct3/4 DE promoter region was changing along differentiation process. HUCBNSC after neural differentiation revealed higher methylation status in promoter region than in undifferentiated cells. Those changes correlate with the expression of Oct3/4 gene. Supported by grant no 0141/P01/2008/35.
3

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

81%
Domanska-Janik K., Habich A., Sarnowska A., Janowski M.
Acta Neurobiologiae Experimentalis
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2006
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tom 66
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nr 4
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 (posttransplantation) properties.
4

Close cell contacts stimulates generation and propagation of neural progenitors from human umbilical cord blood

71%
Habich A., Jurga M., Lukomska B., Barry-Laszewicz U., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2006
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tom 66
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nr 4
5

Freshly isolated and neurally directed human umbilical cord mononuclear cells (HUCB-MNs) exert distinct effects on functional recovery following focal brain damage in the rat

71%
Pawlak E., Janowski L., Habich A., Lukomska B., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
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nr 1
Focal brain damage following stroke leads to severe functional impairments. The aim of the study was to compare therapeutic effectiveness of intra-arterial infusion of HUCB-MNs at different stages of their neural conversion in vitro. Methods: Focal brain damage of dorsolateral striatum was induced in Wistar rats by stereotactic injection of previously established low dose of ouabain (1 μl, 5 mmol). Three days later 107 HUCB-MNs cells were infused (during 3 min) into carotid artery. Thirty days following surgery groups of 7ñ8 rats were housed in large enriched environment cages with various toys. Rats were behaviorally tested for 30 days after lesion. Results: Freshly isolated cells were much more effective in enhancing recovery from motor defi cits measured in walking beam task. Rats treated with HUCB-MNs cells presented also tendency to reduce turning bias and apomorphine induced rotations affected by unilateral lesion. This therapy enhances also recovery from impairments visible in object recognition task. However, rats treated with neurally directed HUCB-MNs also showed a signifi cant improvement in this task. The observed effects were much more prominent in T-maze habit learning task where cell treatment attenuated substantially lesion-induced learning defi cits. What interesting, the mechanism underlying this improvement seems to be different from this observed spontaneously in non-injured animals. Conclusions: Freshly isolated and neurally directed HUCB-MNs differently enhance recovery from distinct functional defi cits induced by focal brain damage. Non-cultured HUCB-MNs seems to be more effective in reducing motor defi cits. Neurally directed HUCB-MNs may be more potent in restoration of impaired habit learning processes. Supported by MSHE grant no 2PO5A05430
6

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

71%
Habich A., Szablwska-Gadomska I., Zayat V., Buzanska L., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr 1
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 umbilical- cord-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.
7

Systemic treatment with freshly isolated HUCB-MN5 cells is the most effective in restoring function following brain demage in rats

61%
Pawlak E., Janowski M., Habich A., Jablonska A., Lukomska B., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2009
|
tom 69
|
nr 3
The aim of the study was to compare therapeutic effectiveness of intra-arterial infusion of human umbilical cord-blood derived mononuclear (HUCB-MNs) cells at different stages of their neural conversion in vitro. Freshly isolated HUCB-MNs (D-0) neuraly directed progenitors (D-3) obtained during 3 days culture of HUCB-MNs and neural stem cells (NSC) line derived from HUCB-MNs were assessed. Focal brain damage of dorsolateral striatum was induced in Wistar rats by stereotactic injection of previously established low dose of ouabain (1 μl or 1,5 μl 5 mmol). Three days later 107 HUCB cells were infused into carotid artery. Following surgery rats were housed in large enriched environment cages, in groups of 7–8 animals per cage, for 30 days. Behavioral assessing consisted of: tests for sensorimotor defi cits (walking beam task, rotarod), habit learning task, exploratory behavior (open fi eld test) and apomorphine induced rotations. Functional effects of different subsets of HUCB cells were diverse in various behavioral tests and hard to conclude which stage of neural conversion of cord blood cells is the most effective in functional recovery. Additional analysis was applied: scores concerning positive effects of cells treatment visible in all parameters were calculated. The sum of scores revealed that the most effective in functional restoration and reduction of lesion volume were freshly isolated D-0 HUCB cells. Supported by Medical Research Centre statutory fund.
8

Generation of stable HEK293 cell lines expressing cell reprogramming factors

61%
Sypecka J., Zayat V., Szablowska-Gadomska I., Habich A., Winiarska H., Buzanska L.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr 1
Induced pluripotent stem cells (iPSCs) are the product of somatic cell reprogramming into an undifferentiated embryonic-like state. These pluripotent cells might adopt various phenotypes by means of bioengineering methods and therefore might serve for disease modeling, pharmaceutical screening and cellular replacement therapies. Transcription factors such as Oct4, Sox2, Klf4 and Myc play the crucial role in the cell converting. The aim of our study was to obtain the protein extracts for the purpose of cell reprogramming experiments. Methods The cells of HEK 293 (Human Embryonic Kidney) line (ATTC/CRL15-73) have been transfected by non-viral, HiFect method with the pCMV cDNA9R-myc plasmid, coding one of the selected factors: Oct4, Sox2 or Klf4. After transfections, cells were cultured in low density for 2-3 weeks in the presence of neomycin to select the resistant (i.e.transfected) colonies. The expression of c-myc as a marker of stable transfectants was determined by western blot analysis. The overexpressed reprogramming proteins were gently extracted with non-denaturating CellLytic buffer supplemented with protease inhibitor cocktail and stored for the future application. Results. Isolation and propagation of an individual cells from neomycin-resistant colonies allowed us to obtain about 20-30 clones for each transcription factor. The c-myc positive clones have been selected for further in vitro culturing with the purpose of continual generation of Oct4, Sox2 or Klf4 proteins. Conclusions. The presented study resulted in successful generation of stable HEK293 cell lines that could express each of the three human reprogramming factors fused with the myc tag and with polyarginine (9R) to facilitate intracellular trafficking. The extracted proteins might therefore be used in induction of cell reprogramming experiment with the aim of generating IPSCs for potential neurorestorative therapies. Supported by grant 5978/B/P01/2010/38 and No N N302 597838.
9

Functional effect of human umbilical cord blood-derived mononuclear cells (HUCB-MNC) transplanted systemically into focal brain injured rats

61%
Pawlak E., Janowski M., Habich A., Jablonska A., Drela K., Lukomska B., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2011
|
tom 71
|
nr 1
Cerebral ischemia causes severe functional deficits due to the death of neuronal and glial cells in the cortex and sub-cortical regions. Stem cell-based therapy could be used to restore lost cells and thus may enhance functional recovery. The aim of the study was to compare therapeutic effectiveness of intra-arterial infusion of human umbilical cord-blood derived mononuclear cells (HUCB-MNC) at different stages of their neural conversion in vitro. Materials and methods. Freshly isolated HUCB-MNC (D-0) neurally directed progenitors (D-3) obtained during 3 days culture of HUCB-MNC and neural-like stem cells (HUCB-NSC) line derived from human cord blood cells were assessed. Focal brain damage was induced in Wistar rats by stereotactic injection of previously established low dose of ouabain into dorsolateral striatum Three days later 107 HUCB cells were infused into internal carotid artery. Following surgery rats were housed in large enriched environment cages, in groups of 7-8 animals per cage, for 30 days observation period. Behavioral assessment consisted of tests for sensorimotor deficits (walking beam task, rotarod, vibrissae elicited forelimb placing), cognitive impairments (habit learning task and object recognition test), exploratory behavior (open field test) and apomorphine induced rotations. At the end of 30 days observation the lesion volume was measured and the presence of donor cells visualized by the expression of mRNA of human reference gene β-2-microglobulin. Results. Functional effects of different subsets of HUCB-MNC treatment shared substantial diversity in various behavioral tests. In walking beam test the most effective in recovery the impaired sensomotor functions in focal brain injured rats were freshly isolated HUCB-MNC (D-0). Also, in rotarod task and in apomorphine induced rotations the tendency to improve scores was observed 30 days following HUCB-MNC (D-0) treatment. In parameters describing open field exploratory behavior the positive effects of HUCB-MNC (D-0) as well as HUCB-NSC cells treatment were observed. However, in cognitive tasks none of tested cell subsets reduced the functional deficits induced by ouabain injection. Thirty days after HUCB cell transplantation we did not observed any mRNA expression of human reference gene in the rat brain samples. Conclusions. Our observation reveals that freshly isolated D-0 HUCB-MNC are the most effective in functional recovery of injured rats. These cells are also the most potent in reducing the ouabain-induced brain lesion volume. The best functional outcome observed after transplantation of HUCB-MNC (D-0) is probably due to the positive effect of therapeutic molecules secreted by these cells than the persistence of donor per se in the host since we did not detect systemically infused human cells in rat brains. Supported by MSHE grant no. 0394/B/P01/2010/38.
10

Transplantation of human umbilical cord blood-derived neural progenitors (HUCB-NP5) improves functional recovery after ouabain lesion in the rat striatum

61%
Gornicka E., Janowski M., Jurga M., Habich A., Wanecka E., Lukomska B.
Acta Neurobiologiae Experimentalis
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2005
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tom 65
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nr 5
11

Human umbilical cord blood derived neural progenitor (HUCB-NP) intra-arterial infusion improves sensorimotor deficits in adult rats with ouabain induced brain iesion

61%
Gornicka E., Janowski M., Habich A., Wanacka E., Lukomska B., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2006
|
tom 66
|
nr 4
12

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

61%
Jezierska-Wozniak K., Barczewska M., Habich A., Wojtacha P., Badowska W., Maksymowicz W., Wojtkiewicz J.
Folia Histochemica et Cytobiologica
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2017
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tom 55
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nr 3
13

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

51%
Gornicka-Pawlak E., Janowski M., Habich A., Jablonska A., Drela K., Kozlowska H., Lukomska B., Sypecka J., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
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 107 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 30th 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.
14

Bystander effect of Human Umbilical Cord Blood Mononuclear Cells administered systemically in long-term repair processes after brain injury

51%
Kozlowska H., Pawlak E., Habich A., Janowski M., Jablonska A., Wanacka E., Lukomska D., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Stem cell transplantation offers an exciting new therapeutic avenue for stroke not only to prevent damage, which has been the focus of conventional therapeutic strategies, but also to actually repair the injured brain. Indeed, exogenous stem cell grafting in animal models of CNS damage improves function by replacing the lost neurons. However, therapeutic mechanism different from the expected contribution of cell replacement have been also postulated. Many studies applying systemic delivery of cells in ischemic stroke disorders have shown significant functional recovery with very few or frequently no cells entering brain. It seems that transplanted cells could propel local micro-environmental signals to sustain active endeavors for damaged neurons substitution. The question arises if systemic infusion of cells enhances endogenous neurogenesis previously activated by focal ischemic brain injury. Materials and methods: Experimental model of focal ischemic brain injury was performed by local application of Na/K ATP-ase pump inhibitor - ouabain (OUA) (1 µl/50nmol) into the striatum of CsA-immunosuppressed adult Wistar rats. Three days later 107 human umbilical cord blood CD34- mononuclear cells (HUCB-MNC) were infused into internal carotic artery. At 30 day thereafter rat brains were removed and the neurogenic regions and tissue around the damaged areas were analyzed immunohistochemically. Results: Analysis of brain tissue in OUA injured rats transplanted with HUCB-MNC revealed augmentation of proliferative cells (Ki-67+) in subventricular zone (SVZ) of ipsilateral hemisphere and at the border of the lesion area as well as higher number of DCX+ cells in SVZ. Moreover, the extensive neuroblast migration and their accumulation in the perinfarct striatum were observed in comparison to non-transplanted rats after OUA injury onset. HUCB-MNC injection into rats with brain infarct showed a significant increase of cells with immature (Nestin+) or more mature (NF-200+) neuronal phenotypes observed in the tissue alongside OUA lesion. The intensive staining of GFAP at the border of injured area in HUCB-MNC transplanted and nontransplanted rats reflected gliosis however, the increased expression of GFAP in brain tissue of the former ones may point to the possible expansion of endogenous progenitors. In conclusions, HUCB-MNC transplanted systemically into OUA focal ischemic brain injured rats activate the endogenous stem cell compartment where the newly arisen cells adopt a neuronal or astrocytic fate. This effect may prove applicable for future clinical therapy. Supported by MSHE grants: 0142/B/P01/2008/35 and 0394/B/ P01/2010/38
15

Serum free culture of 3D scaffold-based aggregates of human neural stem cells derived from cord blood

51%
Jurga M., Sarnowska A., Habich A., Janowski M., Lukomska B., Lipkowski A. W., Kurzepa K., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
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2006
|
tom 66
|
nr 4
16

Analysis of the neurogenic potential of human umbilical cord blood neural stem cells (HUCB-NSC) after their transplantation into rat brain

51%
Kozlowska H., Markiewicz I., Jablonska A., Habich A., Zychowicz M., Wanacka E., Domanska-Janik K., Lukomska B.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
|
nr 1
Transplantation (tx) of neural stem cells (NSC) is the key strategy of cell replacement therapy in the central nervous system. The goal of the study was to compare survival, migration and differentiation of HUCB-NSC after their tx into the brain of neonatal and adult Wistar rats. Methods: HUCB-NSC (2 × 104 ) labeled with CMFDA were tx into SVZ of the postnatal day 0 (P0) rats or into intact brain of adult rats. After 1, 3, 7, 14 or 21 days brains were removed, frozen and cut into 20 μm coronary slices, then immunohistochemical studies were performed to visualize HUCB-NSC fate in the brain. Results: In neonatal rats, 3 days after tx most of HUCB-NSC remained in the graft. During the fi rst week HUCB-NSC started to disperse and migrate. HUCB-NSC situated at the periphery of the graft or in migratory stream display proliferation marker (Ki67). After 7ñ21 days HUCB-NSC survived in the host brain with many cells expressing neuronal or astrocytic phenotypes. Few of HUCB-NSC presented the features of adult neurons (MAP2+ with long protrusions). In adult rats, 3 days after tx, HUCB-NSC form dense deposit with single cells migrating into brain tissue. Most of the HUCB-NSCs stayed undifferentiated with few cells expressing neuronal (NF200) or astrocytic (GFAP) markers. After 7 days numerous HUCB-NSC situated inside the graft underwent degeneration and subsequent depletion. Tx HUCB-NSC induced infl ammatory response detected by macrophage/microglia (ED1+) accumulation and astrogliosis (GFAP+). No viable HUCB-NSC were found after 14 days. Conclusions: Host environment dictates the fate of tx neural stem cells derived from human cord blood however immunological response in adult rats limits the time of observation due to short survival of HUCB-NSC. Supported by MSHE grant no 2PO5A05430
17

MRI-monitored cord blood-derived cell transplantation to the ventricular system of child with global cerebral ischemia-a case report

42%
Janowski M., Kmiec T., Jurkiewicz E., Kropiwnicki T., Habich A., Kotulska K., Jelonek E., Sarnowska A., Litwin M., Boruczkowski D., Lukomska B., Walecki J., Roszkowski M., Jozwiak S., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Introduction: Neurological disorders are the most common cause of serious disability and have a major impact on financial healthrelated burden to society. Most of them are definitely associated with cell death: sudden or chronic. Conventional treatment methods yield disappointing results. Thus the discoveries in stem cell biology have fueled the interest in cell-based therapeutical approach. Based on experimental data cord blood has been proposed as a novel, autologous cell source for pediatric population. Non-invasive monitoring of cell fate following transplantation has been recently recommended as a basis for rational stem cell therapy. Subject: One year old child experienced devastating, cardiac arrest-induced cerebral ischemia. Despite a broad rehabilitation program diagnose of vegetative state has been established three months later. After next three months of continued rehabilitation no noticeable improvement has also been found and the child has been included into study. The protocol has been approved by the ethical commission of The Children’s Memorial Health Institute in Warsaw, Poland. Then the child’s own cord blood cells have been neurally-converted over 10 days in culture within GMP facility. Prior to transplantation cells were labeled with iron oxide (SPIO) for MR imaging. For scaling sensitivity of MR signal different concentrations of SPIO-labeled cells were scanned in the phantom. Then patient received monthly 3 subsequent cell infusions (1.2 x 107 cells each) to lateral ventricles. The follow up continued up to 6 months and included both clinical assessment and MR examinations. Results: High efficiency of neural cell conversion and SPIO labeling as well as no cytotoxicity were observed. The employed method of cell transplantation was found to be efficient to deliver cells to CNS as confirmed by MR imaging. Gradual decrease of SPIO signal intensity was observed over the period of follow up. No adverse events or abnormal reaction to cell implantation was detected. The follow up revealed mild functional improvement - decreased nystagmus, spasticity and the number of epileptic seizures. Moreover, the features of the child contact with parents has appeared, thus vegetative state can not be diagnosed any more. Conclusions: This report indicates that transplantation of autologous, neurally-committed cord blood-derived cells to the ventricular system of child is safe, feasible and able to result with mild functional improvement. Additionally cell-related MRI signal can be monitored for more than 4 months in transplanted brain hemisphere. Supported by MSHE grants no 0141/B/P01/2008/35 and 0142/B/ P01/2008/35.
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