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To optimise the culture conditions for human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs, however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O2). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O2 conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

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.

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version “Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)”. The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility. Refereed/Peer-reviewed

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin - the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds' pore size for neural tissue engineering was 80-100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in "niche"-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.

The umbilical cord blood-derived neural stem/progenitor cells (HUCB-NSCs) potentially represent a rich source of transplantable material for treatment of a wide range of neurological diseases. Although, recently reported effects of their implementation in animal models of brain pathology are still controversial. As a simplified alternative to in vivo transplantation in this work we have applied a long-term organotypic rat hippocampal slice culture (OHC) as a recipient tissue to study bilateral graft/host cells interactions ex vivo. This type of culture can be considered as a kind of reductionistic model of brain transplantation where direct influence of systemic immunological responses to transplanted human cells would be excluded. The transplantation material derived from a HUCB-NSC line developed and characterized in our laboratory and delivered to the slices either as a single-cell suspension or after formation of typical neurospheres in serum-free medium in vitro (N-HUCBs). Experiments were focused on space-temporal context of cell transplantation in relation to their ability to ingrown, migrate, and differentiate within the slice cytoarchitecture. We gain evidences that these responses are strictly dependent on the engraftment site and that cell movement reflects typical routes used for migratory neuroblasts in vivo. The cells implanted at the second week of slice cultivation ingrown readily and deeply into host cytoarchitecture then matured to the level never observed in our transplantation animal models in vivo. Importantly, transplanted neurospheres, in addition to yield exogenous migratory cells to the host tissue can locally inhibit astrocytosis and promote outgrow of DCX-reactive neuroblasts in the surrounding OHC tissue.

Ischemic stroke results in violent impairment of tissue homeostasis leading to severe perturbation within the neurovascular unit (NVU) during the recovery period. The aim of this study was to assess the potential of mesenchymal stem cells (MSCs) originating from Wharton's jelly (WJ) to differentiate into functionally competent cells of endothelial lineage (WJ-EPCs). The protective effect(s) of either primary WJ-MSCs or induced WJ-EPCs was investigated and compared after oxygen–glucose deprivation (OGD) of hippocampal organotypic slices (OHC) in the indirect coculture model. WJ-MSCs, primed in EGM-2 (Lonza commercial medium) under 5% O2, acquired cobblestone endothelial-like morphology, formed capillary-like structures and actively took up DiI-Ac-LDL. Both cell types (WJ-MSCs and WJ-EPCs) were positive for CD73, CD90, CD105, VEGFR-2, and VEGF, but only endothelial-like culture expressed vWF and PECAM-1 markers at significant levels. In the presence of either WJ-MSCs or WJ-EPCs in the compartment below OGD-injured slices, cell death and vascular atrophy in the hypoxia-sensitive CA1 region were substantially decreased. This suggests that a paracrine mechanism may mediate WJ-MSC- and WJ-EPC-dependent protection. Thus, finally, we estimated secretion of the neuro/angio/immunomodulatory molecules IL-6, TGF-β1, and VEGF by these cell cultures. We have found that release of TGF-β1 and IL-6 was TLR ligand [LPS and Poly(I:C)] concentration dependent and stronger in WJ-EPC than WJ-MSC cultures. Simultaneously, the uneven pattern of TLR receptors and modulatory cytokine gene expression was confirmed also on qRT-PCR level, but no significant differences were noticed between WJ-EPC and primary WJ-MSC cultures.

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency.

Objective The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCs CD34- ) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCs CD34- 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-MNCs CD34- 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-MNCs CD34- 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 β-tubulin III, MAP2, GFAP, S100β, 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.

The ability of stem and progenitor cells to proliferate and differentiate into other lineages is widely viewed as a characteristic of stem cells. Previously, we have reported that cells from a CD34(-) (nonhematopoietic) adherent subpopulation of human cord blood can acquire a feature of multipotential neural progenitors in vitro. In the present study, using these cord blood-derived stem cells, we have established a clonal cell line termed HUCB-NSCs (human umbilical cord blood-neural stem cells) that expresses several neural antigens and has been grown in culture for more than 60 passages. During this time, HUCB-NSCs retained their growth rate, the ability to differentiate into neuronal-, astrocyte-, and oligodendrocyte-like cells and displayed a stable karyotype. DNA microarray analysis of HUCB-NSCs revealed enhanced expression of selected genes encoding putative stem and progenitor cell markers when compared to other mononuclear cells. dBcAMP-induced HUCBNSCs were further differentiated into more advanced neuronal cells. This is the first report of the establishment and characterization of a nontransformed HUCB-NSC line that can be grown continuously in a monolayer culture and induced to terminal differentiation. These cells should further our understanding of the regulatory mechanisms involved in NSC self-renewal and differentiation.

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.

Neural stem cell line developed from human umbilical cord blood (HUCB-NSC) [Buzańska et al., 2003. Journal of Neurochemistry 85, 33] is an ethically uncontroversial source of stem cells, able to differentiate into neuronal, astrocytic and oligodendroglial lineages. Developmental fate decisions of HUCB-NSC can be experimentally manipulated in vitro by the presence of trophic factors, mitogenes and neuromorphogenes, but can also be influenced by neurotoxins. In this report two-dimensional (2-D) and three-dimensional (3-D) HUCB-NSC cultures are introduced as useful models for testing developmental neurotoxicity. For 2-D culture models we established a standardized method for the assessment of the growth rate and cell differentiation in 96-well plates. The proliferative capacity of the HUCB-NSC was monitored by the MTT test while their ability to differentiate into neural-like cells by immunocytochemistry of beta-tubulin III and MAP-2 for neurons, GFAP and S-100beta for astrocytes and GalC for oligodendrocytes. The 3-D culture of HUCB-NSC is represented by neurospheres. Proliferation and migration of the intermediate precursors from attached neurospheres are shown to be controlled and altered by various growth factors and further modulated by the extracellular matrix component-fibronectin. Thus, neurospheres derived from the HUCB-NSC line can represent a suitable model of the activation of dormant stem cells residing in their niche, and can be used for neurotoxic studies.

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), gamma-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 (I(K+)). Kir was present on most HUCB-NSCs and HUCB-NSCDs, whereas I(K+) 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.

Paralytic tremor (pt) is a sex-linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents approximately 30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP- and DM-20-specific messenger RNAs. Our results showed that both PLP and DM-20 are affected and that the ratio DM-20/PLP was higher in pt rabbits than in age-matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T-->A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons,astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes — 11% of the total population of differentiating cells.

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% O 2 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 O 2 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% O 2 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% O 2 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.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Here we provide a comprehensive data on the unique features of mesenchymal stem cells (MSCs) which makes them feasible and preferred candidate for cell-based therapy in neurological clinic. From this point of view the most important features of these cells are: (1) availability from autologous sources independently from age of patient; (2) extensive expansion in vitro; (3) immunomodulatory "bystander" function after transplantation in vivo; (4) potentiality to protect, repair or eventually replace impaired or dysfunctional host cells. For complete this last task of functional regeneration of central nervous system, we have to take advantages of MSCs capability for transient, time-locked proliferation, migration to site of injury and their commitment to neuronal differentiation. However, if we are to make progress in the use of MSCs for therapy in the clinic it will be necessary to establish more unified, advanced standards for cells processing in vitro as well as safer and improved procedures for their delivery in vivo.

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.

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.

Physical interactions of cells with the adhesive substrates of the microenvironment as well as the presence of the soluble growth factors are important for the proliferation capacity of neural stem cells. We have used biofunctionalized surface domains microcontact printed with either synthetic polyaminoacid poly-L-lysine or extracellular matrix (ECM) component such as fibronectin, to study the proliferation capacity of human umbilical cord blood-derived neural stem cells (HUCB-NSC). The proliferation measured by the expression of Ki-67 protein was accompanied by the investigation of the cell morphology under the transmission and scanning electron microscopy in different culture time, plating densities of cells and medium condition (serum-free or 2 percent of FBS). The poly-L-lysine domains of defined micro-scale area promoted the presence of round, loosely attached Ki-67-positive cells, while fibronectin domains of the same size allowed appearance of flattened, strongly attached cells with more differentiated phenotype. These results were in agreement with the non-specific, electrostatic type of interaction between cell and substrate on poly-L-lysine and integrin receptor-mediated specific adhesion on fibronectin. In this report we have described in vitro culture conditions, which allow for immobilization of the non-differentiated and highly proliferating population of neural stem/progenitor cells to the biofunctionalized surface. The microarrays with bioactive domains allocating non-differentiated and proliferating neural stem/progenitor cells may find application for drug and chemicals toxicology screening of diverse factors influencing neural development.

Cord blood–derived neural stem cells (NSCs) are proposed as an alternative cell source to repair brain damage upon transplantation. However, there is a lack of data showing how these cells are driven to generate desired phenotypes by recipient nervous tissue. Previous research indicates that local environment provides signals driving the fate of stem cells. To investigate the impact of these local cues interaction, the authors used a model of cord blood–derived NSCs co-cultured with different rat brain–specific primary cultures, creating the neural-like microenvironment conditions in vitro. Neuronal and astro-, oligo-, and microglia cell cultures were obtained by the previously described methods. The CMFDA-labeled neural stem cells originated from, non-transformed human umbilical cord blood cell line (HUCB-NSCs) established in a laboratory. The authors show that the close vicinity of astrocytes and oligodendrocytes promotes neuronal differentiation of HUCB-NSCs, whereas postmitotic neurons induce oligodendrogliogenesis of these cells. In turn, microglia or endothelial cells do not favor any phenotypes of their neural commitment. Studies have confirmed that HUCB-NSCs can read cues from the neurogenic microenvironment, attaining features of neurons, astrocytes, or oligodendrocytes. The specific responses of neurally committed cord blood–derived cells, reported in this work, are very much similar to those described previously for NSCs derived from other “more typical” sources. This further proves their genuine neural nature. Apart from having a better insight into the neurogenesis in the adult brain, these findings might be important when predicting cord blood cell derivative behavior after their transplantation for neurological disorders.

Searching for a reliable source of alternative neural stem cells for experimental treatment of neurological disorders we have established neural stem cell line derived from human umbilical cord blood (HUCB-NSC) (Buzanska et al. 2006). These cells have been shown to differentiate along neuronal and glial lineages in the promoting in vitro conditions. In the current study we transplanted HUCB-NSC into rat brain to determine whether the neural progenitors would be able to survive, migrate and eventually adopt neural phenotypes after exposure to central nervous system (CNS) microenvironment. Our experiments revealed that HUCB-NSC grafting into the brain of adult rats limited their survival up-to two weeks probably due to their elimination by severe immunological host reaction evoked by xenotransplantation. HUCB-NSC graft in neonates survived longer time in rat brain, migrated, proliferated and differentiated into neuronal cells however their presence in the host tissue did not exceed more than five weeks after transplantation.

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.

From the time of discovery that among the cord blood mononuclear cell population there are cells capable of changing their fate towards the neural lineage and producing functional neurons and macroglial cells, our attempts have been focused on the understanding of the underlying mechanism of this transition. We have deciphered the first steps of neural stem/progenitor gene induction in aggregating culture of cord blood mononuclear cells, their rapid phenotypic conversion under the influence of neuromorphogenic signals due to mitogen activation and their ability to expand and develop a prototypic, long-living line with neural stem cell properties. Evidence has accumulated that human umbilical cord-derived and neurally committed cells, due to their capacity for self-renewal, multilineage differentiation, plasticity and ability for long-lasting growth in vitro, provide unique material for the cell therapy of a wide spectrum of neurological diseases. The putative regenerating potential of these cord blood-derived neural stem/progenitor cells was evaluated after transplantation in experimental models of brain injury. In spite of initial promising data, the results indicate an urgent need to improve available animal model protocols in order to increase immuno-tolerance toward transplanted human cells.

Umbilical cord blood can be a rich source of stem/progenitor cells, not only for hematopoetic but also for other tissue-specific lineages. Recently, we have developed a novel, self-renewed neural-like stem cell line named HUCB-NSC from human cord blood. To test if HUCB-NSCs can supply brain in need of regeneration, we injected these cells into immunosuppressed intact rat forebrain and to animals suffering from a photothrombotic cortical lesion at 48 h after injury. The survival, migration, and differentiation of the transplanted HUCB-NSCs were measured at 7 and 30 days post-transplantation by immunohistochemical methods. Results show survival and extensive migration of transplanted neural-like progenitors into damaged brain cortex during the first week of post-stroke recovery. The donor cells accumulated mainly in peri-infarct area and then differentiated showing a strong co-expression of neuronal (NF-200) but only moderate of astrocytic (GFAP) cell markers. However, the paucity of HUCB-NSCs detected within post-ischemic rat brain at the end of a 1 month period, as well as acute rejection of grafted cells by intact, yet cyclosporin A (CsA) immunosuppressed, rat brain tissue, suggests development of a severe adverse host reaction to the presence of alien donor cells and an urgent need for further study of the immunological response evoked by xenotransplantations of human cord blood-derived cells in animal experimental models.

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.

Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.

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.

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.

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.

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.