The long-term safety and efficacy of bilateral transplantation of human fetal striatal tissue in patients with mild to moderate Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease involving progressive motor, cognitive and behavioural decline, leading to death approximately 20 years after motor onset. The disease is characterised pathologically by an early and progressive striatal neuronal cell loss and atrophy, which has provided the rationale for first clinical trials of neural repair using fetal striatal cell transplantation. Between 2000 and 2003, the 'NEST-UK' consortium carried out bilateral striatal transplants of human fetal striatal tissue in five HD patients. This paper describes the long-term follow up over a 3-10-year postoperative period of the patients, grafted and non-grafted, recruited to this cohort using the 'Core assessment program for intracerebral transplantations-HD' assessment protocol. No significant differences were found over time between the patients, grafted and non-grafted, on any subscore of the Unified Huntington's Disease Rating Scale, nor on the Mini Mental State Examination. There was a trend towards a slowing of progression on some timed motor tasks in four of the five patients with transplants, but overall, the trial showed no significant benefit of striatal allografts in comparison with a reference cohort of patients without grafts. Importantly, no significant adverse or placebo effects were seen. Notably, the raclopride positron emission tomography (PET) signal in individuals with transplants, indicated that there was no obvious surviving striatal graft tissue. This study concludes that fetal striatal allografting in HD is safe. While no sustained functional benefit was seen, we conclude that this may relate to the small amount of tissue that was grafted in this safety study compared with other reports of more successful transplants in patients with HD.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880.

Potential role of NF-kappaB in adult neural stem cells: the underrated steersman?

Neural stem cells are precursors of neurons and glial cells. During brain development, these cells proliferate, migrate and differentiate into specific lineages. Recently neural stem cells within the adult central nervous system were identified. Informations are now emerging about regulation of stem cell proliferation, migration and differentiation by numerous soluble factors such as chemokines and cytokines. However, the signal transduction mechanisms downstream of these factors are less clear. Here, we review potential evidences for a novel central role of the transcription factor nuclear factor kappa B (NF-kappaB) in these crucial signal transduction processes. NF-kappaB is an inducible transcription factor detected in neurons, glia and neural stem cells. NF-kappaB was discovered by David Baltimore's laboratory as a transcription factor in lymphocytes. NF-kappaB is involved in many biological processes such as inflammation and innate immunity, development, apoptosis and anti-apoptosis. It has been recently shown that members of the NF-kappaB family are widely expressed by neurons, glia and neural stem cells. In the nervous system, NF-kappaB plays a crucial role in neuronal plasticity, learning, memory consolidation, neuroprotection and neurodegeneration. Recent data suggest an important role of NF-kappaB on proliferation, migration and differentiation of neural stem cells. NF-kappaB is composed of three subunits: two DNA-binding and one inhibitory subunit. Activation of NF-kappaB takes place in the cytoplasm and results in degradation of the inhibitory subunit, thus enabling the nuclear import of the DNA-binding subunits. Within the nucleus, several target genes could be activated. In this review, we suggest a model explaining the multiple action of NF-kappaB on neural stem cells. Furthermore, we discuss the potential role of NF-kappaB within the so-called brain cancer stem cells.

Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate

Adult human neural crest-derived stem cells (NCSCs) are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of human adult inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates. Using minimally-invasive surgery methods isolation is efficient even in older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75(NTR), and S100. Immunoelectron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to nonmyelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 and the neural crest markers Slug and SOX10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28, and NANOG, whereas expression of WDR5, KLF4, and c-MYC was nearly similar. ITSCs were able to differentiate into cells with neuro-ectodermal and mesodermal phenotype. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in severe combined immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75(NTR) positive ITSCs that formed larger neurospheres and proliferated faster than p75(NTR) negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human NCSCs for potential cell-replacement therapy.

Adult craniofacial stem cells: sources and relation to the neural crest

During the process of development, neural crest cells migrate out from their niche between the newly formed ectoderm and the neural tube. Thereafter, they give rise not only to ectodermal cell types, but also to mesodermal cell types. Cell types with neural crest ancestry consequently comprise a number of specialized varieties, such as ectodermal neurons, melanocytes and Schwann cells, as well as mesodermal osteoblasts, adipocytes and smooth muscle cells. Numerous recent studies suggest that stem cells with a neural crest origin persist into adulthood, especially within the mammalian craniofacial compartment. This review discusses the sources of adult neural crest-derived stem cells (NCSCs) derived from the cranium, as well as their differentiation potential and expression of key stem cell markers. Furthermore, the expression of marker genes associated with embryonic stem cells and the issue of multi- versus pluripotency of adult NCSCs is reviewed. Stringent tests are proposed, which, if performed, are anticipated to clarify the issue of adult NCSC potency. Finally, current pre-clinical and clinical data are discussed in light of the clinical impact of adult NCSCs.

Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Origin and regenerative potential of vertebrate mechanoreceptor-associated stem cells

Meissner corpuscles and Merkel cell neurite complexes are highly specialized mechanoreceptors present in the hairy and glabrous skin, as well as in different types of mucosa. Several reports suggest that after injury, such as after nerve crush, freeze injury, or dissection of the nerve, they are able to regenerate, particularly including reinnervation and repopulation of the mechanoreceptors by Schwann cells. However, little is known about mammalian cells responsible for these regenerative processes. Here we review cellular origin of this plasticity in the light of newly described adult neural crest-derived stem cell populations. We also discuss further potential multipotent stem cell populations with the ability to regenerate disrupted innervation and to functionally recover the mechanoreceptors. These capabilities are discussed as in context to cellularly reprogrammed Schwann cells and tissue resident adult mesenchymal stem cells.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro

Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate. © 2015 Wiley Periodicals, Inc.

Lineage-Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage-negative (Lin(-)) cells to test the hypothesis that Lin(-) cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham ( laparotomy only, untreated); Nx (five-sixth nephrectomy and untreated); NxLC1 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy day 15); and NxLC3 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein-1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin-dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin(-) cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin(-) cell-treated rats than in untreated rats. Lin(-) cell treatment significantly improved survival. These data suggest that Lin(-) cell treatment protects against chronic renal failure. STEM CELLS 2009; 27: 682-692

Human Multipotent Mesenchymal Stromal Cells from Distinct Sources Show Different In Vivo Potential to Differentiate into Muscle Cells When Injected in Dystrophic Mice

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Gene Expression Profile of Mesenchymal Stem Cells from Paired Umbilical Cord Units: Cord is Different from Blood

Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use.