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Expression of Neural Markers in Human Umbilical Cord Blood
Abstract
A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.
... Transplanted cells were identified in the brain and investigated for expression of neural marker proteins in vivo, the presence of which would point to the differentiation of umbilical cells to those of neuronal or glial lineages [14,16,17]. Additional in vitro studies have also demonstrated the capability of umbilical cells to adapt neuronal or glial characteristics [18][19][20]. Thus, some crucial questions remain to be answered: Is the proportion of transplanted umbilical cells expressing markers of neural differentiation sufficient to account for the beneficial effects observed in vivo? ...
... After 48 hours, i.e., 2 days in vitro (DIV), nonadherent cells were transferred to a new flask in which those cells were cultured for another 2 days (4 DIV) and became adherent. For cell expansion, the medium was replaced with neural proliferation medium (PM), which consisted of 50% DMEM/50% Ham's F12, supplemented with epidermal growth factor (EGF; 20 ng/mL) and fibroblast growth factor-2 (FGF-2; 20 ng/mL) [20], and cells were cultured for 3 days. After 7 DIV, cells were transferred to neural differentiation medium (DM), in which EGF and FGF-2 were substituted by alltrans retinoic acid (RA; 0.5 mM) and nerve growth factor (NGF; 100 ng/mL), and were cultured for another 7 days (14 DIV). ...
... However, alternative or additional mechanisms like lineage selection cannot be excluded. Incipient differentiation of the adherent mononuclear cell population in vitro was demonstrated by morphological changes as well as by expression of neural marker proteins, which had also been observed previously using different culture protocols [18][19][20]24,28]. However, there is only one report on successful neuronal differentiation that includes demonstration of the electrophysiological properties of these cells in vitro [29]. ...
... The result of present study showed that when the cells treated with EGF display relatively high potency to expand and to grow differentiated colonies and this agrees with Kojima and Tator, (2000) and Sanchez-Ramos et al., (2001) that the role of EGF in the differentiation are: ...
... Our results also observed by Sanchez-Ramos et al., (2001) who demonstrated that RA and nerve growth factor (NGF) exhibit a change in phenotype of these cells to the neuronal cells and expressed molecular markers usually associated with neurons and glial cells. A study carried by Kogler et al., (2005) showed that UCB cells are able to differentiate into neural cells that express neurofilament and the implantation of these cells into the intact rat brain showed migratory activity and typical neuron-like morphology. ...
... The possible applications of the cord blood in the treatment of the neural disorders were highlighted by the first evidence in the early 2000s that cord blood stem cells can cross the tissue and even germ-line barriers to attain in vitro neural features. These pioneering studies came out in parallel from the Labs of Krystyna Domanska-Janik in Warsaw, Poland, and Paul Sanberg in Tampa, USA (Buzanska et al. 2001(Buzanska et al. , 2002Sanchez-Ramos et al. 2001;Zigova et al. 2001). ...
... The pioneering research on the derivation of neural stem cells from hUCB carried out in the group of Paul Sanberg was performed on cryopreserved, nonselected mononuclear cells expanded in the presence of mitogens (EGF and bFGF) and neural induction "N2" medium (Sanchez-Ramos et al. 2001). Differentiation of the expanded cells in the presence of RA and NGF resulted in the increased immunoreactivity of Musashi-1, βIII tubulin, and GFAP in the differentiating population. ...
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.
... Our observation that undifferentiated hDFCs expressed several neural makers by microarray analysis suggesting (15,16). Previous reports have shown that MSI-1 was expressed during early neuronal development of neural stem/progenitor cell cultures from mouse brain and human umbilical cord blood cells (16). ...
... Our observation that undifferentiated hDFCs expressed several neural makers by microarray analysis suggesting (15,16). Previous reports have shown that MSI-1 was expressed during early neuronal development of neural stem/progenitor cell cultures from mouse brain and human umbilical cord blood cells (16). Consistent with the microarray data, these results suggest that hDFCs contain a neural stem/progenitor cell population. ...
Neurotrauma and neurodegenerative diseases are associated with the loss of functioning neural cells in the nervous system. Many studies reported that function can be restored by replacing lost cells with stem cells that can mature into neural cells. From this perspective,mesenchymal stem cells represent a valuable tool for regenerative therapy because of their ability to differentiate along several lineages, such as adipocytes, osteoblasts, chondrocytes and neural cells. The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells(hDFCs)have the capacity to commit to differentiation into multiple cell types. In this study, we investigated the capacity of hDFCs to differentiate into neural cells, and the efficiency of the neural differentiation process.
There was a gene relevant to a neural cell in hDFC. We expanded these findings to address the gene expression of neural markers in hDFCs during neuronal differentiation. The expression levels of Musashi(MSI)-1 and -2, which are neural progenitor cell markers,microtubule-associated protein 2 (MAP2) which is a neuronal cell marker, and glial fibrillary acidic protein (GFAP)and myelin basic protein (MBP), which are glial cell markers, were up-regulated in hDFCs undergoing neural differentiation during culture in neuronal differentiation medium. The expression of tubulin-β-III(TUBB3), which is an early neuronal cell marker, was peaked on day 3. Furthermore, expression of Nestin(NES) did not change. In conclusion, these in vitro data suggest that hDFCs have the capacity to differentiate along neural lineages, raising the possibility that hDFCs may represent a practical and convenient source of adult stem cells for cell-based therapies to treat neurological diseases or trauma.
... When exposed to nerve growth factor and RA, the derived umbilical cells produce progeny that show positivity of neural and glial cells markers. However, biology of the cells is currently poorly understood, and it is likely that positive effects of these cells are related to their neurotrophic action, rather than actual neuronal circuitry formation [22,23]. ...
... IL-6 preconditioning of neural stem cells was found to induce secretion of VEGF from these stem cells through activation of signal transducer and activation of transcription. Platelets also contribute to tumor induced angiogenesis as platelets are the carrier of angiogenic growth factors including VEGF [31,23]. Certain indirect angiogenic cytokines such as TGF-β1, may act via induction of bFGFs and VEGF gene expression in the cells resident near endothelial cells in vivo. ...
... Cells collected from the umbilical cord blood are a promising source of cells for grafting. Some reports have shown that the umbilical cord blood contains cells that may express neuronal markers (Buzanska, Machaj, Zablocka, Pojda, & Domanska-Janik, 2002;Sanchez-Ramos et al., 2001). The umbilical cord is also a rich source of hematopoietic and mesenchymal stem cells (Wang et al., 2004) and has compelling potential for therapeutic use in neurological diseases due to the ability of these cells to differentiate into neural lineages (Buzanska et al., 2002;Jeong et al., 2004;Li et al., 2004;McGuckin, Forraz, Allouard, & Pettengell, 2004;Rogers & Casper, 2004;Sanchez-Ramos et al., 2001). ...
... Some reports have shown that the umbilical cord blood contains cells that may express neuronal markers (Buzanska, Machaj, Zablocka, Pojda, & Domanska-Janik, 2002;Sanchez-Ramos et al., 2001). The umbilical cord is also a rich source of hematopoietic and mesenchymal stem cells (Wang et al., 2004) and has compelling potential for therapeutic use in neurological diseases due to the ability of these cells to differentiate into neural lineages (Buzanska et al., 2002;Jeong et al., 2004;Li et al., 2004;McGuckin, Forraz, Allouard, & Pettengell, 2004;Rogers & Casper, 2004;Sanchez-Ramos et al., 2001). ...
The last few years have seen a growing interest in stem cell research. The media has helped to keep attention to the subject making it as popular as therapeutic cloning. A number of studies highlighted the importance of stem cells as prospective therapy for neurodegenerative conditions. The possibility of successful treatment for such pathologies using stem cells has raised enormous hope. Whilst in some countries biotechnologists have realised the commercial potential of the field, human stem cell research and its clinical applications have constantly been subject of ethical and legal debate. The present review brings an overview of recent aspects of stem cell research for central nervous system disorders, with special attention to Parkinson's and Alzheimer's diseases, cerebral ischemic insult and spinal cord injury. Furthermore, the ethical implications regarding the use of embryonic stem cells as a fundamentally more promising cell type are discussed.
... DNA microarray analysis shows that MSCs grown in the presence of RA and NGF exhibit significant changes in the expression of 322 genes out of a total of 12,600 human genes represented on the DNA chip. And at least 20 can be bound to products found in developing neurons, glia, or neural cells[116].Locatelli et al. induced the formation of spheres from murine bone marrow ...
Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells present multiple characteristics that make them candidates to be applied in cell therapy for neurodegenerative diseases, such as their secretory action, migration to the lesion area, and immunomodulatory potential. These cells have a high capacity for mesodermal differentiation; however, numerous studies have shown that MSCs can also differentiate into neurons. However, despite positive results in multiple trials in which undifferentiated MSCs transplanted into animal models of neurodegenerative diseases, some studies suggest that the therapeutic effects obtained are enhanced by the use of MSCs differentiated towards the neuronal lineage before transplant. In this sense, there are several methods to induce in vitro reprogramming of MSCs towards the neu-ronal lineage, including chemical substances, growth factors, cocultures with neural lineage cells, transfection of genes, miRNAs, etc., and small molecules stand out. Therefore, this article compares multiple experimental tests in which these inducers promote neuronal differentiation of MSCs and identify those methods that originate an optimal neuronal differentiation. The analysis includes the percentage of differentiation, maturation, expression of neuronal markers, functionality, and cell survival considering the intrinsic characteristics of the MSCs used as the tissue of origin and the species from which they were isolated.
... The second cell source was the progenitor cells found in human umbilical cord blood (hUCB) which could also be induced to express neural markers Sanchez-Ramos et al., 2001). In studies of stroke, we found that animals treated with hUCB 24 h after ischemic injury had good functional recovery and reduced infarct size (Chen et al., 2001;Vendrame et al., 2004;Willing et al., 2003). ...
The American Society for Neural Therapy and Repair (ASNTR) started 30 years ago in 1993 as the American Society for Neural Transplantation (ASNT), with an emphasis on neural transplantation. Through the years, the Society has been shaped as much by our expanding knowledge of neurodegenerative disorders and how to treat them as it has by politics and culture. What once felt like a leash on neuroscience research, has turned into an advantage as neural transplantation evolved into neural therapy and repair. This brief commentary provides a personalized account of our research during the Society's years.
... Non-cryopreserved autologous volume-and red blood cellreduced umbilical cord blood (UCB) cells (Cotten et al., 2014) were administered with TH in one study. UCB cells are adult stem cells derived from the human umbilical cord, and have been shown in vitro to express various marker proteins for early neural precursors, as well as neurons, astrocytes and oligodendrocytes (Sanchez-Ramos et al., 2001;Rosenkranz and Meier, 2011;Ballen et al., 2013). UCB cells also secrete chemokines, cytokines and growth factors including BDNF and VEGF. ...
Objective: Therapeutic hypothermia (TH) is the current standard of care for neonatal hypoxic-ischemic encephalopathy (HIE), yet morbidity and mortality remain significant. Adjuvant neuroprotective agents have been suggested to augment hypothermic-mediated neuroprotection. This analysis aims to identify the classes of drugs that have been used in combination with hypothermia in the treatment of neonatal HIE and determine whether combination therapy is more efficacious than TH alone.
Methods: A systematic search of PubMed, Embase and Medline from conception through December 2022 was conducted. Randomized- and quasi-randomized controlled trials, observational studies and retrospective studies evaluating HIE infants treated with combination therapy versus TH alone were selected. Primary reviewers extracted information on mortality, neurodevelopmental impairment and length of hospitalization for meta-analyses. Effect sizes were pooled using a random-effects model and measured as odds ratio (OR) or mean difference (MD) where applicable, and 95% confidence intervals (CI) were calculated. Risk of bias was assessed using the tool from the Cochrane Handbook for Systematic Reviews of Interventions.
Results: The search strategy collected 519 studies, 16 of which met analysis inclusion criteria. HIE infants totaled 1,288 infants from included studies, 646 infants received some form of combination therapy, while 642 received TH alone. GABA receptor agonists, NMDA receptor antagonists, neurogenic and angiogenic agents, stem cells, glucocorticoids and antioxidants were identified as candidate adjuvants to TH that have been evaluated in clinical settings compared to TH alone. Length of hospitalization was significantly reduced in infants treated with combination therapy (MD −4.81, 95% CI [−8.42. to −1.19], p = .009) compared to those treated with TH alone. Risk of mortality and neurodevelopmental impairment did not differ between combination therapy and TH alone groups.
Conclusion: Compared to the current standard of care, administration of neuroprotective adjuvants with TH reduced the duration of hospitalization but did not impact the risk of mortality or neurodevelopmental impairment in HIE infants. Meta-analysis was limited by a moderate risk of bias among included studies and small sample sizes. This analysis highlights the need for preclinical trials to conduct drug development studies in hypothermic settings to identify relevant molecular targets that may offer additive or synergistic neuroprotection to TH, and the need for larger powered clinical trials to determine the dose and timing of administration at which maximal clinical benefits are observed for adjuvant neuroprotectants.
... In 2001, Sanberg et al. published two papers that would outline the next decade of research. They found that cells from human umbilical cord blood (HUCBC) could develop neural characteristics, expressing markers of glia and neurons when exposed to retinoic acid (RA) and nerve growth factor (NGF) (Sanchez-Ramos et al., 2001). In addition, HUCBC could be injected intravenously to promote recovery in an animal stroke model rather than intracranially, a much more invasive and risky procedure (Chen et al., 2001). ...
It has been more than thirty years since the two inaugural IBNS presidents sat down at a larger neuroscience conference and decided that there should be more to behavioral neuroscience than a single theme at a meeting. The progeny of these conversations is the International Behavioral Neuroscience Society (IBNS) and this year will be its thirty year anniversary. We reflect back on the last thirty years of the research career of the society's second president, Paul R. Sanberg, as an example of how behavioral neuroscience research has changed these last few decades.
... DNA microarray analysis shows that MSCs grown in the presence of RA and NGF exhibit significant changes in the expression of 322 genes out of a total of 12,600 human genes represented on the DNA chip. And at least 20 can be bound to products found in developing neurons, glia, or neural cells[116].Locatelli et al. induced the formation of spheres from murine bone marrow ...
Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells have a high capacity for mesodermal differentiation; however, numerous studies have shown that MSCs can also differentiate into cells of endodermal and ectodermal lineages. Due to this multilineage differentiation capacity, these cells could function as restoratives of various cell populations after transplantation. However, not only their differentiation potential makes them ideal candidates for this, but also a series of trophic properties that promote regeneration in the surrounding tissue, such as their migratory capacity, secretory and immunomodulatory actions. This review analyzes several MSC transplantation trials to treat neurological diseases such as demyelinating injury, spinal cord injury, paraplegia, Parkinson's disease, cochlear injury, and Alzheimer's disease. These cells could facilitate functional recovery in multiple models of neurodegenerative diseases and nervous system injuries by using their trophic capacities, reducing inflammation in the injured area, reducing apoptosis, and enhancing endogenous neurogenesis through the secretion of bioactive factors. Furthermore, since cells derived from patients have demonstrated disease-associated differences in various brain diseases, these cells represent an excellent candidate for the study of these diseases, functioning as "a window to the brain."
... Transplanted human CB cells have been observed to migrate to the site of injury within 24 h [44]. Mechanisms for the neuroprotective effect of CB stem cells include differentiation into neurons, astrocytes, oligodendrocytes, and microglia [45][46][47][48][49]; antiapoptotic and anti-inflammatory actions [50]; stimulation of angiogenesis; production of trophic factors [51,52]; and mitochondrial donation [53,54]. Endothelial progenitor cells decrease neuroinflammation and cell apoptosis [55]. ...
Hypoxic-ischaemic encephalopathy (HIE) is a severe complication of asphyxia at birth. Therapeutic hypothermia, the standard method for HIE prevention, is effective in only 50% of the cases. As the understanding of the immunological basis of these changes increases, experiments have begun with the use of cord blood (CB) because of its neuroprotective properties. Mechanisms for the neuroprotective effects of CB stem cells include antiapoptotic and anti-inflammatory actions, stimulation of angiogenesis, production of trophic factors, and mitochondrial donation. In several animal models of HIE, CB decreased oxidative stress, cell death markers, CD4+ T cell infiltration, and microglial activation; restored normal brain metabolic activity; promoted neurogenesis; improved myelination; and increased the proportion of mature oligodendrocytes, neuron numbers in the motor cortex and somatosensory cortex, and brain weight. These observations translate into motor strength, limb function, gait, and cognitive function and behaviour. In humans, the efficacy and safety of CB administration were reported in a few early clinical studies which confirmed the feasibility and safety of this intervention for up to 10 years. The results of these studies showed an improvement in the developmental outcomes over hypothermia. Two phase-2 clinical studies are ongoing under the United States regulations, namely one controlled study and one blinded study.
... DNA microarray analysis shows that MSCs grown in the presence of RA and NGF exhibit significant changes in the expression of 322 genes out of a total of 12,600 human genes represented on the DNA chip. And at least 20 can be bound to products found in developing neurons, glia, or neural cells[116].Locatelli et al. induced the formation of spheres from murine bone marrow ...
Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells present multiple characteristics that make them candidates to be applied in cell therapy for neurodegenerative diseases, such as their secretory action, migration to the lesion area, and immunomodulatory potential. These cells have a high capacity for mesodermal differentiation; however, numerous studies have shown that MSCs can also differentiate into neurons. However, despite positive results in multiple trials in which undifferentiated MSCs transplanted into animal models of neurodegenerative diseases, some studies suggest that the therapeutic effects obtained are enhanced by the use of MSCs differentiated towards the neuronal lineage before transplant. In this sense, there are several methods to induce in vitro reprogramming of MSCs towards the neu-ronal lineage, including chemical substances, growth factors, cocultures with neural lineage cells, transfection of genes, miRNAs, etc., and small molecules stand out. Therefore, this article compares multiple experimental tests in which these inducers promote neuronal differentiation of MSCs and identify those methods that originate an optimal neuronal differentiation. The analysis includes the percentage of differentiation, maturation, expression of neuronal markers, functionality, and cell survival considering the intrinsic characteristics of the MSCs used as the tissue of origin and the species from which they were isolated.
... The first, by Woodbury et al. [5], applied simple chemical compounds as media additives to induce differentiation. The second method, by Sanchez-Ramos et al., [9], utilises cytokine and growth factor media additives. Interestingly the two methods vary considerably for the time the MSCs need to spend in culture before showing the phenotypic changes that indicate differentiation. ...
The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation.
... Nucleated umbilical cord blood (UCB) cells have been identified as pluripotent stem cells, as they could differentiate into cells with characteristics of neurons, astrocytes, oligodendrocytes and microglia in vitro. [351][352][353] Transplantation of human UCB cells stimulates tissue repair and cognitive recovery in a dose-dependent way after neonatal H/I brain injury. 354 The human UCB cells improve neurological functions by preventing apoptosis, suppressing inflammation, enhancing angiogenesis and neurogenesis. ...
With the emergence of novel technologies and ground breaking concepts in BBB basic research in the past decades, the researchers’ attention on the critical role of blood brain barrier (BBB) integrity after stroke is surging. The main novelty of this review is that it not only highlights recent findings of the signaling pathways mediating ischemic BBB disruption, such as vascular permeability, lactate transportation failure, mitochondrial crisis and ion channel and water channel disability, endothelial actin polymerization, junctional proteins disassembly and endothelial transcytosis. Evolutional breakthroughs in identification of novel molecules that orchestrates the neuro-vascular unit restoration, immune cells of distinct phenotype that play dualistic roles in the evolution of ischemic brain injury are also included in this review. The aim of this review is to provide intriguing therapeutic targets to protect the BBB after cerebral ischemia.
... Even though there are many protocols available which, mainly, use DMSO and β-mercaptoethanol, as well as interaction between different cell lineages [37], best results for induction of neural differentiation were obtained using a protocol based on the use of neural growth factor (NGF), brain-derived neural factor (BDNF), basic fibroblast growth factor (bFGF), dibutyril cAMP (dbcAMP), isobutylmethylxanthine (IBMX) and indomethacin [38][39][40]. The medium used was DMEM/F-12 (70:30 v/v), supplemented with B-27 and N2, as it is usually done when growing neural stem cells. ...
Adipose stromal cells are promising tools for clinical applications in regeneration therapies, due to their ease of isolation from tissue and its high yield; however, their ability to transdifferentiate into neural phenotypes is still a matter of controversy. Here, we show that combined chemical and neurotrophin stimulation resulted in neuron-like morphology and regulated expression and activity of several genes involved in neurogenesis and neurotransmission as well as ion currents mediated by NMDA and GABA receptors. Among them, expression patterns of genes coding for kinin-B1 and B2, α7 nicotinic, M1, M3 and M4 muscarinic acetylcholine, glutamatergic (AMPA2 and mGlu2), purinergic P2Y1 and P2Y4 and GABAergic (GABA-A, β3-subunit) receptors and neuronal nitric oxide synthase were up-regulated compared to levels of undifferentiated cells. Simultaneously, expression levels of P2X1, P2X4, P2X7 and P2Y6 purinergic and M5 muscarinic acetylcholine receptors were down-regulated. Agonist-induced activity levels of the studied receptor classes also augmented during neuronal transdifferentiation. Transdifferentiated cells expressed high levels of neuronal β3-tubulin, NF-H, NeuN and MAP-2 proteins as well as increased ASCL1, MYT1 and POU3F2 gene expression known to drive neuronal fate determination. The presented work contributes to a better understanding of transdifferentiation induced by neurotrophins for a prospective broad spectrum of medical applications.
... Human umbilical cord blood also provides a rich source of adult stem cells and has therefore generated much interest in stroke, traumatic brain injury, and SCI. Generally, MSCs transplantation following SCI leads to amelioration of inflammation, apoptosis, and glial scarring in conjunction with increased axonal regrowth, angiogenesis, and tissue sparing in both cervical [110][111][112][113][114][115][116][117] and thoracic models [106,[118][119][120][121][122][123][124][125][126][127][128][129][130][131][132]. ...
Cervical-level injuries account for the majority of presented spinal cord injuries (SCIs) to date. Despite the increase in survival rates due to emergency medicine improvements, overall quality of life remains poor, with patients facing variable deficits in respiratory and motor function. Therapies aiming to ameliorate symptoms and restore function, even partially, are urgently needed. Current therapeutic avenues in SCI seek to increase regenerative capacities through trophic and immunomodulatory factors, provide scaffolding to bridge the lesion site and promote regeneration of native axons, and to replace SCI-lost neurons and glia via intraspinal transplantation. Induced pluripotent stem cells (iPSCs) are a clinically viable means to accomplish this; they have no major ethical barriers, sources can be patient-matched and collected using non-invasive methods. In addition, the patient’s own cells can be used to establish a starter population capable of producing multiple cell types. To date, there is only a limited pool of research examining iPSC-derived transplants in SCI—even less research that is specific to cervical injury. The purpose of the review herein is to explore both preclinical and clinical recent advances in iPSC therapies with a detailed focus on cervical spinal cord injury.
... UCBSC exhibit significant trophic effects and have demonstrated neural protective effects in rodent models of neonatal hypoxia-ischemia [23][24] . UCBSC may also be capable of direct fate conversion to other lineages such as CD34 + hematopoietic and endothelial progenitor cells (~1%) [25] which can secrete angiogenic factors to promote neurogenesis [26] , neural and glia-like cells that may be directly used for cell replacement therapy in ND [27] . ...
... It has been shown that Lin − cells are capable of generating a high number of neuroblasts after in vitro induction in defined serum-free media (Jurga et al. 2012). Nevertheless, only a few groups have demonstrated that human UCB cells give rise to neural progenitors in vitro (Sanchez-Ramos et al. 2001, Buzanska et al. 2002, Habich and Domanska-Janik 2011, Jurga et al. 2012). In addition to such in vitro studies, Schwarting and colleagues (2008) using MCAO mouse model has demonstrated significant decrease in infarct volume and apoptosis after about 72 hours of intravenous transplantation of Lin − cells. ...
Umbilical cord blood (UCB)-derived stem/progenitor cells (SPCs) have demonstrated the potential to improve neurologic function in different experimental models. SPCs can survive after transplantation in the neural microenvironment and indu ce neuroprotection, endogenous neurogenesis by secreting a broad repertoire of trophic and immunomodulatory cytokines. In this study, the influence of brain-derived neurotrophic factor (BDNF) pre-treatment was comprehensively evaluated in a UCB-derived lineage-negative (Lin-) SPC population. UCB-derived Lin- cells were evaluated with respect to the expression of (i) neuronal markers using immunofluorescence staining and (ii) specific (TrkB) receptors for BDNF using flow cytometry. Next, after BDNF pre-treatment, Lin- cells were extensively assessed with respect to apoptosis using Western blotting and proliferation via BrdU incorporation. Furthermore, NT-3 expression levels in Lin- cells using RQ PCR and antioxidative enzyme activities were assessed. We demonstrated neuronal markers as well as TrkB expression in Lin- cells and the activation of the TrkB receptor by BDNF. BDNF pre-treatment diminished apoptosis in Lin- cells and influenced the proliferation of these cells. We observed significant changes in antioxidants as well as in the increased expression of NT-3 in Lin- cells following BDNF exposure. Complex global miRNA and mRNA profiling analyses using microarray technology and GSEA revealed the differential regulation of genes involved in the proliferation, gene expression, biosynthetic processes, translation, and protein targeting. Our results support the hypothesis that pre-treatment of stem/progenitor cells could be beneficial and may be used as an auxiliary strategy for improving the properties of SPCs.
... Es konnte gezeigt werden, dass Stammzellen aus Nabelschnurblut in unterschiedliche Gewebe wie Blut-, Herzmuskel-, Knochen-, Leber-, Bauchspeicheldrüsen-oder Nervenzellen differenzieren können [7,9,19,22,41,46,49]. Hieraus ergeben sich zahlreiche potentielle medizinische Anwendungen, beispielsweise für die Behandlung des Myokardinfarkts oder neurologischer Krankheitsbilder wie etwa des Morbus Parkinson. ...
... Similar kind of study on hUCB non-haematopoietic multipotent stem cells showed differentiation of these cells into cortical GABAergic neurons with upregulated expression of GABAergic regulatory enzymes and transcription factors namely, MASH1 and DLX1 & 2 [86]. Even unspecified mononuclear cells derived from hUCB have capability to express neuronal markers namely, Musashi-1 and TUJ-1 and GFAP under the influence of specific growth factors/ neurotrophins [113]. Seo and Cho have observed that differentiation of MSCs into neuronal cells induced the secretion of numerous trophic factors which can modulate different cellular processes such as neurogenesis, inflammation, angiogenesis and apoptosis [114]. ...
The developing brain is found highly vulnerable towards the exposure of different environmental chemicals/drugs, even at concentrations, those are generally considered safe in mature brain. The brain development is a very complex phenomenon which involves several processes running in parallel such as cell proliferation, migration, differentiation, maturation and synaptogenesis. If any step of these cellular processes hampered due to exposure of any xenobiotic/drug, there is almost no chance of recovery which could finally result in a life-long disability. Therefore, the developmental neurotoxicity (DNT) assessment of newly discovered drugs/molecules is a very serious concern among the neurologists. Animal-based DNT models have their own limitations such as ethical concerns and lower sensitivity with less predictive values in humans. Furthermore, non-availability of human foetal brain tissues/cells makes job more difficult to understand about mechanisms involve in DNT in human beings. Although, the use of cell culture have been proven as a powerful tool for DNT assessment, but many in vitro models are currently utilizing genetically unstable cell lines. The interpretation of data generated using such terminally differentiated cells is hard to extrapolate with in vivo situations. However, human umbilical cord blood stem cells (hUCBSCs) have been proposed as an excellent tool for alternative DNT testing because neuronal development from undifferentiated state could exactly mimic the original pattern of neuronal development in foetus when hUCBSCs differentiated into neuronal cells. Additionally, less ethical concern, easy availability and high plasticity make them an attractive source for establishing in vitro model of DNT assessment. In this review, we are focusing towards recent advancements on hUCBSCs-based in vitro model to understand DNTs.
Background:-Human umbilical cord blood- mesenchymal stem cells(HUCB-MSCs) hold great promise invitro neural differentiation and therapy for neurodegenerative disorders. It has demonstrated that the numberand differentiating potential of bone marrow mesenchymal stem cells (MSCs) decrease with age. Therefore,the search for alternative sources of MSCs is of significant value.Objective:-To determine the possibility of obtaining clonally expanded UCB-MSCs and evaluate their rapidinduction and differentiation into neural cells in vitro.Methods:-MSCs were isolated from HUCB by combining gradient density centrifugation with plasticadherence. In order to identify factors that able to lead to neural differentiation, the cultured cells weretreated with retinoic acid (RA) and B-mercaptoethanol (BME). Differentiating characterization of UCBMSCswere detected by immunocytochemistry analysis.Results:-UCB-MSCs appeared like the fibroblast cell and these cells were extensively expanded in culture.The results showed that UCB-MSCs were positive for CD71and CD90 but were negative for CD34. Theimmunocytochemistry staining indicated that the differentiated cells give positive response for nestinemarker. The result confirmed for neural progenitor differentiation of MSCs.Conclusion: The results of this study confirmed that UCB provides a great source of stem cells for using intreatment of neurodegenerative disorders of the central nervous system.
Objective:
To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells (HUCMSCs) to differentiate into neuron- like cells after induction with B27- supplemented serum- free medium.
Methods:
HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium (SCM) (group A), SCM supplemented with 20 ng/mL nerve growth factor (NGF) and 10 ng/mL basic fibroblast growth factor (bFGF) (group B), serum-free medium (SFM) (group C), or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF. The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope. The cell markers were analyzed with flow cytometry and the expressions of nestin, neuron- specific enolase (NSE), neurofilament heavy polypeptide (NEFH), and glial fibrillary acidic protein (GFAP) were quantified by quantitative real-time PCR (qRT-PCR) and Western blotting.
Results:
Before induction, HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29 (99.5%), CD44 (49.6%) and CD105 (77.7%). Neuron-like cells were observed in the cultures on days 7, 10, and 14, and the cell differentiation was the best in group D, followed by groups C, B and A. In all the 4 groups, the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively. The expressions of GFAP, NEFH, nestin and NSE were significantly different between group A and the other 3 groups (P < 0.001 or 0.05).
Conclusions:
B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron- like cells, and the supplementation with cytokines (NGF and bFGF) strongly promotes the cell differentiation.
Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III β-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.
In vitro growth of hematopoietic cells depends on the presence of hematopoietic cytokines. To date, it is unclear if these cells would be able to respond to non-hematopoietic cytokines. In the present study, we have explored this by culturing human hematopoietic cells in presence of neurogenic cytokines. Lineage-negative (Lin⁻) umbilical cord blood (UCB)-derived cells -enriched for hematopoietic stem and progenitor cells- were cultured in presence of different combinations of hematopoietic cytokines, neurotrophins, epidermal growth factor, fibroblast growth factor, and neurogenic culture media, in a 3-phase culture system. A proportion (1–22%) of Lin⁻ UCB hematopoietic cells normally express neural markers and are capable of responding to neural cytokines. Neural cytokines did not have effects on hematopoietic cell proliferation; however, we observed generation of neural-like cells, assessed by morphology, and a significant increase in the proportion of cells expressing neural markers. Such neural-like cells, however, retained expression of hematopoietic markers. It seems that under our culture conditions, no actual transdifferentiation of hematopoietic cells into neural cells occurred; instead, the cells generated in culture seem to be hematopoietic cells that acquired neural features upon contact with neurogenic factors. The identity of UCB cells that acquired a neural phenotype is still unclear.
Microglia-mediated neuroinflammation is one of the most significant features in a variety of central nervous system (CNS) disorders such as traumatic brain injury, stroke and many neurodegenerative diseases. Microglia become polarized upon stimulation. The two extremes of the polarization are the neuron-destructive pro-inflammatory M1-like and the neuron-regenerative M2-like phenotypes. Thus, manipulating microglial polarization towards the M2 phenotype is a promising therapeutic approach for CNS repair and regeneration. It has been reported that nanoparticles are potential tools for regulating microglial polarization. Gold nanoclusters (AuNCs) could penetrate the blood-brain barrier and have neuroprotective effects, suggesting the possibility of utilizing AuNCs to regulate microglial polarization and improve neuronal regeneration in CNS. In the current study, AuNCs functionalized with dihydrolipoic acid (DHLA-AuNCs), an antioxidant with demonstrated neuroprotective roles, were prepared and their effects on polarization of a microglial cell line (BV2) were examined. DHLA-AuNCs effectively suppressed pro-inflammatory processes in BV2 cells by inducing polarization towards the M2-like phenotype. This was associated with a decrease in reactive oxygen species and reduced NF-kB signaling, and an improvement in cell survival coupled with enhanced autophagy and inhibited apoptosis. Conditioned medium from DHLA-AuNC-treated BV2 cells was able to enhance neurogenesis in both the neuronal cell line N2a and in an ex vivo brain slice stroke model. The direct treatment of brain slices with DHLA-AuNCs also ameliorated stroke-related tissue injury and reduced astrocyte activation (astrogliosis). This study suggests that by regulating neuroinflammation to improve neuronal regeneration, DHLA-AuNCs could be a potential therapeutic agent in CNS disorders.
Objective
This study aimed to use a systematic approach to evaluate the current utilization, safety, and effectiveness of cell therapies for neurological diseases in human. And review the present regulations, considering United States (US) as a representative country, for cell transplantation in neurological disease and discuss the challenges facing the field of neurology in the coming decades.
Methods
A detailed search was performed in systematic literature reviews of cellular-based therapies in neurological diseases, using PubMed, web of science, and clinical trials. Regulations of cell therapy products used for clinical trials were searched from the Food and Drug Administration (FDA) and the National Institutes of Health (NIH).
Results
Seven most common types of cell therapies for neurological diseases have been reported to be relatively safe with varying degrees of neurological recovery. And a series of regulations in US for cellular therapy was summarized including preclinical evaluations, sourcing material, stem cell manufacturing and characterization, cell therapy product, and clinical trials.
Conclusions
Stem cell-based therapy holds great promise for a cure of such diseases and will value a growing population of patients. However, regulatory permitting activity of the US in the sphere of stem cells, technologies of regenerative medicine and substitutive cell therapy are selective, theoretical and does not fit the existing norm and rules. Compiled well-defined regulations to guide the application of stem cell products for clinical trials should be formulated.
Introduction:
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord. Treatment options are limited due to the complexity of underlying disease factors. Cell therapy, using human umbilical cord blood (hUCB) cells may be a promising new treatment for ALS, mainly by providing a protective microenvironment for motor neuron survival. Areas covered: Composition and in vitro and in vivo differentiation of hUCB cells and the advantages of cord blood as a source of transplant cells are discussed. A brief history of hUCB in treatment of an ALS animal model and the feasibility of these cells in therapy for ALS patients is provided. Current ALS clinical trials are also deliberated. Expert opinion: Among multiple advantages, hUCB cells' production of various anti-inflammatory/growth/trophic factors makes them an attractive cell source for ALS therapy. Biodistribution and optimal hUCB cell dose for transplantation have been determined in preclinical studies. Repeated intravenous cell doses during disease progression may be the best approach for cell-based ALS treatment. Accumulated evidence shows the efficacy of naïve or genetically modified MNC hUCB cells in the treatment of ALS and provide a superior basis for the development of clinical trials in the near future.
The term ‘cellular therapy’ identifies a modality of medical treatment in which drugs are replaced with cells. Examples of classical cellular therapies that have been successfully used for decades include bone marrow or peripheral blood stem cell transplantation and red blood cell or platelet transfusion. Currently, the terms ‘cellular therapy’ are mostly used to identify rather sophisticated procedures in which well defined cell sub-populations undergo some degree of manipulation or engineering, such as positive and negative cell selection, exvivo expansion, gene modification, etc., under strict environmental control ensured in laboratory facilities known as ‘cell factories’.
Umbilical cord blood (UCB) is an alternative source of hematopoietic progenitor and stem cells to treat many malignant and nonmalignant disorders. This pioneering finding started with several in vitro studies that led to the first hematopoietic cell transplantation in which UCB was used instead of bone marrow. Since then the idea to preserve UCB for future use became appealing. Thereafter, many CB banks(CBBs) have been established and over the last decade it became a popular option worldwide. The full process of UCB banking includes donor recruitment, UCB collection, processing, testing, cryopreservation, storage, listing, search, selection, reservation, release, and distribution for administration. In this chapter I will highlight the first discoveries in the field of UCB, the main steps to preserve this highly enriched source of stem cells and its most established clinical applications.
This review represents the findings, reflecting the main achievements in the scope of cell based therapy of nervous diseases and therapeutic potential of cord blood cells in experimental and clinical neurology. The cord blood-derived cell populations are classified and the possibility of their application in therapy of neurological deficit is demonstrated. Different approaches to cord blood cell transplantation are considered, depending on administration way and cell concentration. Possible mechanisms of therapeutic effect of these cells, as well as their ability to migrate into the brain through the blood-brain barrier are discussed.
Neural stem cells (NSC) exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. A great deal of literature has addressed the identification, screening and isolation of neural stem cells. The NSC in these regards have been reviewed (Gage, 1994; Gage et al., 1995b; Weiss et al., 1996b; McKay et al., 1997; Vescovi & Snyder, 1999; Gage, 2000). The mammalian adult brain reserves a limited pool of neural stem cells, which can differentiate and incorporate into the mature brain. In this chapter, the regulation of stem cells in the adult brain and spinal cord is discussed. The potency and neurogenesis of endogenous NSC in a genetically predetermined state are frequently affected by environmental factors, such as enriched environment, excercise, stress, or stroke. Neural replenishment, a process preserved in lower animals, is now being assessed in the repair of the mammalian nervous system. In addition, NSCcan also be recruited from non-neural origins. The mechanisms that convert early stem cells or non-NSCinto NSCremain elusive, but are beginning to be unveiled. Potential uses of neural stem cells, including transplantation to repair missing cells and activation of endogenous cells to promote “self-repair” are also reviewed in this chapter.
In light of recent research findings, the transplantation of umbilical cord blood stem cells offers a novel cell-based delivery system for gene therapy in brain tumors. This chapter presents the role of naive cord blood stem cells in glioma therapy. There are many advantages of human umbilical cord blood as a source of mesenchymal stem cells (MSC) as compared to bone marrow or adipose tissue. First, the collection of cord blood is easy and painless and the cord blood can be stored for later use. Second, human umbilical cord blood–derived mesenchymal stem cells (hUCBSC) are more primitive than MSC isolated from other tissue sources. Third, hUCBSC have a higher proliferative capacity and a faster population doubling time that remains unaltered after 30 passages. Finally, hUCBSC has lower immunogenicity and graft-versus-host reactivity when compared to bone marrow stromal cells.
Although initial in vitro evidence pointed to the differentiation of human cord blood cells (HUCBCs) into neuronal and glial lineages, transplantation of these cells never resulted in terminally differentiated neurons. This raised the suspicion that the beneficial effect of HUCBCs in models of central nervous system (CNS) disorders and injury may be attributable to alternative biologic properties. The indication that HUCBCs may have anti-inflammatory and immunoregulatory properties has recently emerged from animal studies.
During consecutive fetal development stages, many specific stem cell populations arise, expand, migrate, and differentiate, participating in the formation of the fetus, umbilical cord, and placenta. Some of them may be found only in the organs of developing fetus; others are present in cord blood or reside in umbilical cord and placenta. Collection of cells from fetuses is ethically questionable, and the material from aborted fetuses is heterogeneous both for its biological (poor cell viability, presence of pathogens) and genetic qualities. The only sources of fetal stem and progenitor cells, not causing technical or ethical problems, are cord blood, umbilical cord, and placenta, being the “biological waste material” after newborn’s delivery. The developmental age of cells present in cord blood, and even more cells residing in umbilical cord and placenta tissues, may vary – some of them may be the remnants of the cells migrating at the early stages of fetus development, residing in the tissues in dormant, noncycling state.
Overview Over the past two decades, hematopoietic stem-cell transplantation has emerged as an effective approach to curative therapy for both pediatric and adult patients with aggressive or recurrent malignancies, congenital immunodeficiency diseases, some genetic diseases, including inborn errors of metabolism and hemoglobinopathies, and congenital and acquired bone-marrow-failure syndromes. Traditionally, autologous or allogeneic stem and progenitor cells have been obtained from bone marrow or mobilized peripheral blood. More recently, banked umbilical-cord blood (UCB) has emerged as an alternative source of stem and progenitor cells for transplantation. Cord blood is readily available and can be transplanted across partially mismatched human leukocyte antigen (HLA) barriers, increasing the availability of allogeneic stem-cell donors for patients lacking traditional HLA-matched related and unrelated donors. Additional applications of stem-cell therapies envisioned in the next decade, including regenerative therapies for nonhematopoietic tissues damaged by injury or disease, are currently unproven and the subject of ongoing preclinical research. A major limitation to stem-cell transplantation therapy is donor availability. Only 20-25% of patients in need of a transplant will have an HLA-matched relative who can serve as their donor. Of those lacking a related donor, approximately 25% of Caucasian patients will identify an HLA-matched unrelated living bone-marrow donor through the National Marrow Donor Program and other donor registries, but less than 10% of patients of ethnic minority backgrounds will find a suitably matched unrelated marrow donor. For the remaining patients, a fully matched unrelated stem-cell donor cannot be identified. © Cambridge University Press 2005 and Cambridge University Press, 2009.
The search for a cure for intractable diseases and the hope for replacing defective tissues with healthy or younger ones has been the driving force for stem cell research. Therapeutic cloning using embryonic stem cells seems to be the most versatile approach. Adult stem cells could also be manipulated for purposes of gene therapy or drug delivery. This necessitates a certain degree of controlled delivery, in order to prevent uncontrolled cell division that may result in tumor formation. Finding stem cell or other cells that have immunosuppressive qualities, or that facilitate engraftment of other co-transplanted tissues, is another dynamic research direction. The most promising candidates are mesenchymal stem cells from bone marrow and cord blood, and recently, from isolated Sertoli cells. Finding and isolating the cells of interest is perhaps the most active field of investigation, and ironically the most disappointing. The dramatic potential of cell therapy involves replacing diseased cells and a diseased environment with healthy cells and a healthy environment comprised more accessory cells in addition to a myriad of growth and trophic factors. This intricate composition initiates a cascade of events that involve release of intrinsic factors and stimulation of healthy cells to proliferate and initiate the desired process of repair. This premise, along with the advantages of high stability and non-tumorigenicity of cord blood cells compared to embryonic stem cells, warrants the search for the fountain of youth in the young cells of the cord blood.
Overview. Over the past several decades, hematopoietic stem cell transplantation has emerged as an effective approach to curative therapy for both pediatric and adult patients with aggressive or recurrent malignancies, congenital immunodeficiency diseases, some genetic diseases, including inborn errors of metabolism and hemoglobinopathies, and congenital and acquired bone marrow failure syndromes. Traditionally, autologous or allogeneic stem and progenitor cells have been obtained from bone marrow or mobilized peripheral blood. More recently, banked umbilical cord blood (UCB) has emerged as an alternative source of stem and progenitor cells for transplantation. Cord blood is readily available and can be transplanted across partially mismatched human leukocyte antigen (HLA) barriers, increasing the availability of allogeneic stem cell donors for patients lacking traditional HLA-matched related and unrelated donors. Additional applications of stem cell therapies envisioned in the next decade, including regenerative therapies for non-hematopoietic tissues damaged by injury or disease are currently unproven and the subject of ongoing preclinical and clinical research. A major limitation to stem cell transplantation therapy is donor availability. Only 20%-25% of patients in need of a transplant will have an HLA-matched relative who can serve as their donor. Of those lacking a related donor, approximately 25% of Caucasian patients will identify an HLA-matched unrelated living bone-marrow donor through the National Marrow Donor Program and other donor registries, but less than 10% of patients of ethnic minority backgrounds will find a suitably matched unrelated marrow donor. For the remaining patients, a fully matched unrelated stem cell donor cannot be identified.
Objective: To explore whether neuron-like cells in human umbilical cord blood mononuclear cells (HUBMNCs). Methods: The mononuclear cells were separated from 30 newborns umbilical cord blood. The expressions of nestin and neuron-specific enolase (NSE) were observed by immunofluorescent staining method. HUBMNCs were cultured in vitro, and the changes of the cells morphology were observed with inverted phase contrast microscope. Results: There were (2. 67 ± 1. 29) nestin positive cells and (7. 37 ±2.46) NSE positive cells in each high power field (HP, ×200) of HUBMNCs by immunofluorescent staining. There were 3-5/HP neuron-like cells which were large and with a plurality of coarse long protrusions similar to dendrites and axons at cultured 10th - 12th d. Conclusion: There are a few neuron-like cells in HUBMNCs.
Regenerative therapy against stroke is a novel concept. According to recent progress in stem cell biology, adult neurogenesis was rediscovered and a neural stem cell was able to be cultured in vitro. There are still a lot of problems to be clarified to regulate neurogenesis and transplantation. We can now start to use these materials to improve neurological findings after stroke.
The developing brain is vulnerable towards compounds interfering with processes explicitly involved in brain development. These processes include neural stem/progenitor cell (NS/PC) proliferation, migration, differentiation, apoptosis as well as myelination, synaptogenesis, and network formation. Due to specific actions of compounds on these processes, which differ from the most common mechanisms of adult neurotoxicity, specific developmental neurotoxicity (DNT) testing is necessary. In view of the large societal impact that only small changes in IQ pose on a population and the financial burdens associated with this drop in societal IQ, the assessment of adverse effects of chemicals on the highly complex process of brain development is even more indispensable. In this chapter, recent developments on human stem/progenitor cell-based assays for DNT testing are reviewed. Thereby, distinct available cell sources as well as diverse culturing methods of these cells are described. Their applications for compound testing are portrayed and finally a summary and brief recommendation on the future of DNT testing by using stem/progenitor cells in vitro is given.
Umbilical cord blood is a readily available source of hematopoietic stem cells used with increasing frequency as an alternative to bone marrow or peripheral stem cells for transplantation in the treatment of malignant and nonmalignant conditions in children and adults. The recent interest in stem cell research and public fascination with promises of stem cell-based therapies, fueled by the media, have led researchers to explore the potential of UCB stem cells in therapy for regenerative medicine applications.
Human umbilical cord blood was collected from full-term deliveries scheduled for cesarean section. Mononuclear cells were isolated, amplified and induced as mesenchymal stem cells. Isolated mesenchymal stem cells tested positive for the marker CD29, CD44 and CD105 and negative for typical hematopoietic and endothelial markers. Following treatment with neural induction medium containing brain-derived neurotrophic factor for 7 days, the adherent cells exhibited neuron-likecellular morphology. Immunohistochemical staining and reverse transcription-PCR revealed that theinduced mesenchymal stem cells expressed the markers for neuron-specific enolase andneurofilament. The results demonstrated that human umbilical cord blood-derived mesenchymalstem cells can differentiate into neuron-like cells induced by brain-derived neurotrophic factor invitro.
Over the last few decades, there has been an explosion in stem cell research. The investigation of umbilical cord blood (UCB) cells as a treatment for stroke is even more recent. Ease of collection and the ability to maintain their stem cell properties post-cryopreservation made these cells very attractive candidates for treatment development initially. UCB cells have many advantages including a wide variety of cell types present, including hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells, lymphocytes, and monocytes, which enhances their ability to modulate multiple targets impacted by neurodegenerative processes. Although the precise mechanisms of action are still being researched, UCB cells have been shown to benefit functional recovery and also reduce infarct size post-stroke. They have also demonstrated an ability to provide these benefits when administered peripherally and within 24–48 h post-stroke, which immensely expands the current treatment window of 3–4 h for tissue plasminogen activator. This chapter highlights the current research with UCB cells in the development of a novel treatment for stroke and demonstrates the great therapeutic potential of these cells.
Stroke causes irreversible and permanent damage in the brain immediately adjacent to the region of reduced blood perfusion. During a stroke, every second means the death of 32,000 neurons, that is, 1.9 million cells every minute. In that same minute, the brain loses 14 billion synapses and 7.5 miles of myelinated fibers. Regenerative immediate action is fundamental. Currently, the only effective treatment for stroke, tissue-plasminogen activator, has a very narrow therapeutic window. These disappointing outcomes clearly solicit developing any therapeutic modality, especially cell-based therapy. Human umbilical cord blood (HUCB) cells, because of their primitive nature and ability to differentiate into various nonhematopoietic cells, including neural cells, may be useful as an alternative target for cell-based therapies requiring either the replacement of lost cells and/or substitution of missing substances. Over the past several years, our team and others have investigated the therapeutic potential of mononuclear cells found within cord blood for stroke and other neurological disorders. We have demonstrated that systemic administration of HUCB cells provides significant benefit in stroke models of neural degeneration. Interestingly, it is more likely that the indirect neurotrophic effects from these cells such as release of various growth or antiinflammatory factors affording neural protection and promoting immunomodulatory benefits may be the underlying mechanism rather than direct neural replacement. These protective and restorative effects may prove critical to preserving tissue integrity over the course of a stroke.
The cDNA for the developmentally regulated, neurite outgrowth-promoting protein HB-GAM (heparin-binding growth-associated molecule) was recently cloned and shown to encode a novel lysine-rich sequence that is homologous with retinoic acid-induced sequences suggested to function in cell differentiation (Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724). The same sequence was found for the mitogenic and neurite outgrowth-promoting protein pleiotrophin (Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). In this study, we have constructed a recombinant baculovirus using the cDNA that encodes the putative preprotein of HB-GAM. The putative secretion signal of HB-GAM is cleaved off in the baculovirus expression system, and the recombinant protein is rapidly secreted to the culture medium. Recombinant HB-GAM purified from the culture medium retains the biochemical characteristics and the neurite outgrowth-promoting activity found for the tissue-derived protein. Studies on the neurite outgrowth-promoting activity suggest that HB-GAM functions as an extracellular matrix-associated protein that enhances axonal growth in perinatal cerebral neurons of the rat. Since the same predicted amino acid sequence has been ascribed to a mitogenic protein, mitogenic activities of the recombinant HB-GAM and of tissue-derived HB-GAM fractions were also studied. Recombinant HB-GAM did not display any significant mitogenic activity, suggesting that tissue-derived HB-GAM preparations may contain other heparin-binding mitogenic factors. We identified in brain-derived HB-GAM fractions a 17-kDa protein (p17) that is detached from heparin by a slightly higher salt concentration as compared to HB-GAM. We suggest that p17 is structurally distinct from HB-GAM and responsible for the mitogenic actions of tissue-derived HB-GAM fractions.
Neurons are highly polarized cells that extend long processes, the axons and dendrites, to form contacts with target cells. The formation and maintenance of this specialized morphology relies on the assembly of an organized microtubule array that is the predominant component of the neuronal cytoskeleton. During this process there is an evolution in the composition and dynamics of microtubules, resulting in stable microtubule bundles that provide structural support and function in intracellular transport along the axon. In this essay we provide an overview of the mechanisms regulating the synthesis and assembly of tubulin in differentiating neurons with particular attention to the roles of multiple tubulin isotypes, posttranslational modifications of tubulin, and microtubule-associated proteins. We conclude that, ultimately, the developmental regulation of microtubules in neurons may require the coordinated expression and posttranslational modifications of tubulin and microtubule-associated proteins to provide biochemical forms that favour specific interactions, each combination conferring distinctive dynamic and functional properties.
Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells. Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS-1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino-terminal region of the large T antigen, which encompasses the Rb-binding domain. Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb. We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression. Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo. In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.
Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.
Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and post-translational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III beta-tubulin (also known as beta6-tubulin). Both the upregulation and the post-translational processing of class-III beta-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III beta-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-beta6, specific to unphosphorylated class-III beta-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III beta-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta6 bound unphosphorylated soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-beta6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-beta6 +ve/TuJ-1 -ve cells); a differentiation stage (U-beta6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III beta-tubulin immunostaining levels. These investigations also conclude that U-beta6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.
Neuronal microtubules have unique stability properties achieved through developmental regulation at the expression and posttranslational levels on tubulins and microtubule associated proteins. One of the most specialized tubulins specific for neurons is class-III beta-tubulin (also known as beta6-tubulin). Both the upregulation and the post-translational processing of class-III beta-tubulin are believed to be essential throughout neuronal differentiation. The present investigation documents the temporal and spatial patterns of class-III beta-tubulin expression throughout neurogenesis. For this study a novel polyclonal antiserum named U-beta6, specific to unphosphorylated class-III beta-tubulin has been developed, characterized and compared with its commercial homologue TuJ-1. Our experiments indicate that the two antibodies recognize different forms of class-III beta-tubulin both in vitro and in vivo. Biochemical data revealed that U-beta6 bound unphosphorylated soluble class-III beta-tubulin specifically, while TuJ-1 recognized both the phosphorylated and unphosphorylated forms of the denatured protein. In vivo U-beta6 was associated with neurogenesis and labelled newly committed CNS and PNS neuroblasts expressing neuroepithelial cytoskeletal (nestin and vimentin) and surface markers (the anti-ganglioside supernatant, A2B5 and the polysialic acid neural adhesion molecule, PSA-NCAM), as well as differentiating neurons. These studies with U-beta6 illustrate three main developmental steps in the neuronal lineage: the commitment of neuroepithelial cells to the lineage (U-beta6 +ve/TuJ-1-ve cells); a differentiation stage (U-beta6 +ve/TuJ-1 +ve cells); and, finally, neuronal maturation correlating with a drop in unphosphorylated class-III beta-tubulin immunostaining levels. These investigations also conclude that U-beta6 is an earlier marker than TuJ-1 for the neuronal lineage in vivo, and it is thus the earliest neuronal lineage marker known so far.
In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form 'neurospheres' in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(-) cells, which included Talpha1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(-). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.
Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family
of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three
neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We
have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns;
by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express
NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated
calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself,
but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration
of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly
effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.
Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.
Research on neural stem cells isolated from embryonic, fetal and adult tissues has engendered novel perspectives regarding the identity, origin and full therapeutic potential of tissue-specific stem cells. Review of the similarities and differences between the stem cells that give rise to nervous system (neuropoiesis) and those that generate the various blood cell lineages (hematopoiesis) provides new insights into the process of neuronal differentiation. In this review, we compare hematopoiesis and neuropoiesis from several perspectives starting with ontogenetic considerations and ending with discussion of the cellular interactions of marrow-derived cells with neural cells in vitro and in vivo.
Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.
The expression pattern described here is that of the chick BMP-1/Tolloid family of secreted metalloproteinases during early stages of development. BMP-1/Tolloid transcripts are expressed in the blastoderm, at gastrulation stages and as the neural plate forms and neural tube folds, BMP-1/Tolloid is found at the neural plate/ectodermal transition. Expression is maintained in the premigratory neural crest, and transiently in the migrating cephalic neural crest cells. BMP-1/Tolloid is also expressed in the caudal, but not in the anterior notochord, and in the ventral neural tube at the time of dorso-ventral patterning. Further sites of BMP-1/Tolloid expression are the lateral plate mesoderm and the dermotome and the myotome of the somites.
This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowth-promoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days E12 and E14 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/D1. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance.
GAP43 has long been regarded as a neurone specific molecule present intraneuronally in both the central and peripheral nervous system, especially during development and regeneration. GAP43 has, however, recently been demonstrated in developing muscle cells of the chicken. In the prsent investigation, we have used immunohistochemistry to investigate whether GAP43 is also expressed in developing human muscle cell. Using specific monoclonal antibodies as markers for developing muscle cells (desmin) and axon terminals (synaptophysin), our results show that GAP43 is expressed in aneural, human embryonic muscle cells.
We have used a combination of immunofluorescence microscopy, northern blotting, ELISA, and isoelectric focusing to characterize the expression of neuronal Class III beta-tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway by retinoic acid. Following 48 h differentiation, beta-III tubulin mRNA is evident and beta-III tubulin appears in the mitotic spindle of neuroblasts. Neurite outgrowth is obvious by day 3, and beta-III tubulin protein and mRNA levels increase concurrently until approximately day 7, when beta-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic beta-III tubulin isoforms appear during neuronal differentiation. The expression of these isoelectric variants occurs concomitant with a temporal increase in the levels of beta-III tubulin present in the colchicine-stable microtubules. These results implicate posttranslational modifications of beta-III tubulin in the increased microtubule stability noted in differentiating P19 neurons.
Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS domain protein (NPAS) 1 and NPAS2, are members of the basic helix-loop-helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain. In situ hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human NPAS1 gene was mapped to chromosome 19q13.2-q13.3, and the mouse Npas1 gene to chromosome 7 at 2 centimorgans. Similarly, the human NPAS2 gene was assigned to chromosome 2p11.2-2q13, and the mouse Npas2 gene to chromosome 1 at 21-22 centimorgans. The chromosomal regions to which human NPAS1 and NPAS2 map are syntenic with those containing the mouse Npas1 and Npas2 genes, indicating that the mouse and human genes are true homologs.
In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens
cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of
cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in
vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting
cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results
focus attention on how information in external stimuli is translated into the number and types of differentiated cells in
the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding
of the origin and stability of cell type in the central nervous system.
The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.
Bone marrow progenitor cells have been shown to contribute to a small proportion of cells in nonhematopoietic tissues including the brain. In the acute unilateral middle cerebral artery occlusion model in spontaneously hypertensive rats following male-to-female bone marrow transplantation, we present data suggesting that 55% more marrow-derived cells, in general, and 161% more GFAP-positive astrocytes, in particular, migrate preferentially to the ischemic cortex than to the contralateral non-ischemic hemisphere. In addition to their biological significance, our findings could have therapeutic implications. Marrow-derived progenitor cells could potentially be used as vehicles for ex vivo gene transfer to the brain.
Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple treatment protocol induced the stromal cells to exhibit a neuronal phenotype, expressing neuron-specific enolase, NeuN, neurofilament-M, and tau. With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF-M. The refractile cell bodies extended long processes terminating in typical growth cones and filopodia. The differentiating cells expressed nestin, characteristic of neuronal precursor stem cells, at 5 hr, but the trait was undetectable at 6 days. In contrast, expression of trkA, the nerve growth factor receptor, persisted from 5 hr through 6 days. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Human marrow stromal cells subjected to this protocol also differentiated into neurons. Consequently, adult marrow stromal cells can be induced to overcome their mesenchymal commitment and may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurologic diseases.
We tested the hypothesis that transplantation of bone marrow stromal cells (MSCs) into the spinal cord after a contusion injury promotes functional outcome. Rats (n = 31) were subjected to a weight driven implant injury. MSCs or phosphate buffered saline was injected into the spinal cord 1 week after injury. Sections of tissue were analyzed by double-labeled immunohistochemistry for MSC identification. Functional outcome measurements using the Basso-Beattie-Bresnehan score were performed weekly to 5 weeks post-injury. The data indicate significant improvement in functional outcome in animals treated with MSC transplantation compared to control animals. Scattered cells derived from MSCs expressed neural protein markers. These data suggest that transplantation of MSCs may have a therapeutic role after spinal cord injury.
FGF2 is a crucial mitogen for neural precursor cells in the developing cerebral cortex. Heparan sulfate proteoglycans (HSPGs) are thought to play a role in cortical neurogenesis by regulating the action of FGF2 on neural precursor cells. In this article, we present data indicating that glypican-4 (K-glypican), a GPI-anchored cell surface HSPG, is involved in these processes. In the developing mouse brain, glypican-4 mRNA is expressed predominantly in the ventricular zone of the telencephalon. Neither the outer layers of the telencephalic wall nor the ventricular zone of other parts of the developing brain express significant levels of glypican-4, with the exception of the ventricular zone of the tectum. In cultures of E13 rat cortical precursor cells, glypican-4 is expressed in cells immunoreactive for nestin and the D1.1 antigen, markers of neural precursor cells. Glypican-4 expression was not detected in early postmitotic or fully differentiated neurons. Recombinant glypican-4 produced in immortalized neural precursor cells binds FGF2 through its heparan sulfate chains and suppressed the mitogenic effect of FGF2 on E13 cortical precursor cells. The spatiotemporal expression pattern of glypican-4 in the developing cerebral wall significantly overlaps with that of FGF2. These results suggest that glypican-4 plays a critical role in the regulation of FGF2 action during cortical neurogenesis.
After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived
cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed
a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual
cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton
neurofilament, and class III β-tubulin) and were able to activate the transcription factor cAMP response element–binding protein
(CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates
a remarkable plasticity of adult tissues with potential clinical applications.
To elucidate the mechanisms underlying physiological development and neurodegenerative disorders of the human brain, information about molecular cell biology of human neurons is indispensable. Necdin, which is expressed in postmitotic neurons, binds to viral oncoproteins and the cell-cycle-related transcription factors E2F and p53. Ectopic expression of necdin in proliferative cells suppresses cell division. Necdin is expressed in neurons in phylogenetically old brain areas such as the brain stem and hypothalamus. The human necdin gene, which resides in the chromosome 15q11-q12 region, is not expressed in the Prader-Willi syndrome, suggesting that necdin is responsible for the pathogenesis of this genomic-imprinting-related neurobehavioral disorder. The Alzheimer amyloid precursor protein (APP) is a membrane-bound protein that is abundantly expressed in postmitotic neurons. The proteolytic processing of APP generates A beta, which is deposited in the brains of patients with Alzheimer's disease. APP is strongly expressed in neurons in phylogenetically new brain areas such as human association cortices. When APP is overexpressed in postomitotic neurons differentiated from human embryonal carcinoma by adenovirus-mediated gene transfer, it induces typical apoptosis through caspase-3 activation. Thus APP may be a proapoptotic molecule involved in neuronal death in Alzheimer's disease.
Direct in vivo gene transfer to the central nervous system (CNS) using recombinant lentiviral vectors (rLV) has emerged as a powerful technique to overexpress various genes of interest in different neuronal populations. This interest is exemplified by the increasing number of studies using rLV vectors to evaluate therapeutic proteins to correct disorders of the nervous system (1–3) or to explore a protein’s involvement in normal function or pathological processes (4). Compared to conventional trangenic techniques for overexpression, rLVs have several attractive features. For example, the level of transgene expression in cells transduced with a rLV is typically higher than obtained with knock-in techniques and is present already at 3–4 d after injection in vivo (5). Second, the expression may be directed to specific regions or cell populations depending on the anatomical location of the rLV injections as well as the design of the vector system. This may be advantageous for the study design since it allows unilateral overexpression, thereby creating an internal control in functional and morphological studies. Finally, it is considerably less time consuming than creating transgenic mouse lines.
Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes
- J Flax
- S Aurora
- C Yang
- C Simonin
- A Wills
- L Billinghurst
- M Jendoubi
- R Sidman
- J Wolfe
- S Kim
- E Snyder
Flax, J., S. Aurora, C. Yang, C. Simonin, A. Wills, L. Billinghurst, M. Jendoubi, R. Sidman, J. Wolfe, S. Kim, and E. Snyder.
1998. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat.
Biotechnol. 16: 1033-1039.
Stem cells in the nervous system
- McKay
Tubulin synthesis and assembly in differentiating neurons
- Laferriere
Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes
- Flax