Instruct: Given a scientific claim, retrieve relevant documents that support or refute the claim.
Query: Pure neural progenitor cell (NPC) populations can only be obtained from cell cultures that undergo passaging, filtration, or other isolation and cell sorting methods.
Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.
Neurogenesis occurs in the hippocampus of the developing and adult brain due to the presence of multipotent stem cells and restricted precursor cells at different stages of differentiation. It has been proposed that they may be of potential benefit for use in cell transplantation approaches for neurodegenerative disorders and trauma. Prolonged release of interleukin-1β (IL-1β) from activated microglia has a deleterious effect on hippocampal neurons and is implicated in the impaired neurogenesis and cognitive dysfunction associated with aging, Alzheimer's disease and depression. This study assessed the effect of IL-1β on the proliferation and differentiation of embryonic rat hippocampal NPCs in vitro. We show that IL-1R1 is expressed on proliferating NPCs and that IL-1β treatment decreases cell proliferation and neurosphere growth. When NPCs were differentiated in the presence of IL-1β, a significant reduction in the percentages of newly-born neurons and post-mitotic neurons and a significant increase in the percentage of astrocytes was observed in these cultures. These effects were attenuated by IL-1 receptor antagonist. These data reveal that IL-1β exerts an anti-proliferative, anti-neurogenic and pro-gliogenic effect on embryonic hippocampal NPCs, which is mediated by IL-1R1. The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.
Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.
Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin(-) MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin(+) cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP(+) Pdgfrα(-) cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation.
Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.
Isolated pure human beta cells would be helpful for a number of research purposes. However, lack of beta cell-specific surface antigens has been a major problem. We aimed to develop a simple method for human beta cell isolation based on the initial elimination of ductal cells by their expression of carbohydrate antigen 19-9 (CA19-9), followed by positive selection of beta cells by their expression of polysialic acid–neural cell adhesion molecule (PSA-NCAM). Cell type-specific expression of CA19-9, NCAM and PSA-NCAM was studied in sections of adult human pancreas and in cultured primary endocrine and exocrine cells. Dispersed human islet cells were purified in two steps, after 4 days of suspension culture, by binding to magnetic microbeads coupled to antibodies against CA19-9 and PSA-NCAM. NCAM expression was detected in ducts and islets in the human pancreas. In contrast, PSA-NCAM immunoreactivity was detected only in islets. PSA-NCAM staining in dispersed cells revealed that the marker is expressed in all endocrine cell types, but not in duct cells. Purification of dispersed islet cells using PSA-NCAM microbeads alone did not completely eliminate contaminating duct cells. However, elimination of the duct cells by CA19-9 microbeads followed by positive sorting of the PSA-NCAM-positive cells in five consecutive islet preparations resulted in 90 to 98% pure endocrine cells, of which 89 to 97% were beta cells. We describe a simple and reproducible method for purification of viable human pancreatic beta cells devoid of exocrine acini and ducts.
New neurons are generated in the adult hippocampus throughout life by neural stem/progenitor cells (NSCs), and neurogenesis is a plastic process responsive to external stimuli. We show that canonical Notch signaling through RBP-J is required for hippocampal neurogenesis. Notch signaling distinguishes morphologically distinct Sox2(+) NSCs, and within these pools subpopulations can shuttle between mitotically active or quiescent. Radial and horizontal NSCs respond selectively to neurogenic stimuli. Physical exercise activates the quiescent radial population whereas epileptic seizures induce expansion of the horizontal NSC pool. Surprisingly, reduced neurogenesis correlates with a loss of active horizontal NSCs in aged mice rather than a total loss of stem cells, and the transition to a quiescent state is reversible to rejuvenate neurogenesis in the brain. The discovery of multiple NSC populations with Notch dependence but selective responses to stimuli and reversible quiescence has important implications for the mechanisms of adaptive learning and also for regenerative therapy.
Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.
We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.
Somatic stem cells have been identified in multiple adult tissues. Whether self-renewal occurs symmetrically or asymmetrically is key to understanding long-term stem cell maintenance and generation of progeny for cell replacement. In the adult mouse brain, neural stem cells (NSCs) (B1 cells) are retained in the walls of the lateral ventricles (ventricular-subventricular zone [V-SVZ]). The mechanism of B1 cell retention into adulthood for lifelong neurogenesis is unknown. Using multiple clonal labeling techniques, we show that the vast majority of B1 cells divide symmetrically. Whereas 20%-30% symmetrically self-renew and can remain in the niche for several months before generating neurons, 70%-80% undergo consuming divisions generating progeny, resulting in the depletion of B1 cells over time. This cellular mechanism decouples self-renewal from the generation of progeny. Limited rounds of symmetric self-renewal and consuming symmetric differentiation divisions can explain the levels of neurogenesis observed throughout life.
Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.
Some authors claim that microglia originate from the neuroepithelium, although most now believe that microglial cells are of mesodermal origin, and probably belong to the monocyte/macrophage cell line. These cells must enter the developing central nervous system (CNS) from the blood stream, the ventricular space or the meninges. Afterward microglial cells are distributed more or less homogeneously through the entire nervous parenchyma. Stereotyped patterns of migration have been recognized during development, in which long-distance tangential migration precedes radial migration of individual cells. Microglial cells moving through the nervous parenchyma are ameboid microglia, which apparently differentiate into ramified microglia after reaching their definitive location. This is supported by the presence of cells showing intermediate features between those of ameboid and ramified microglia. The factors that control the invasion of the nervous parenchyma, migration within the developing CNS and differentiation of microglial cells are not well known. These phenomena apparently depend on environmental factors such as soluble or cell-surface bound molecules and components of the extracellular matrix. Microglial cells within the developing CNS are involved in clearing cell debris and withdrawing misdirected or transitory axons, and presumably support cell survival and neurite growth.
The recent derivation of pluripotent stem cell lines from a number of different sources, including reprogrammed adult somatic cells, raises the issue of the developmental equivalence of these different pluripotent states. At least two different states representing the epiblast progenitors in the blastocyst and the pluripotent progenitors of the later gastrulating embryo have been recognized. Understanding the initial developmental status of the different pluripotent lines is critical for defining starting conditions for differentiation toward therapeutically relevant cell types.
BACKGROUND Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell properties in vitro. DESIGN, SETTINGS, AND PARTICIPANTS The colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, alpha2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18). RESULTS AND LIMITATIONS Five out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133(+) cells was detected. CD133(+) cells; however, like DU145 BCRP(+) (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed alpha2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells. CONCLUSIONS These results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.
Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand- or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.
The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent ‘mesenspheres’ that can self-renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or β3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.
INTRODUCTION The hostile environment after spinal cord injury (SCI) can compromise effects of regenerative therapies. We hypothesized that optimizing the post-traumatic environment with QL6 self-assembling peptides (SAPs) before neural precursor cell (NPC) transplantation would improve cell survival, differentiation and functional recovery. METHODS A total of 90 Wistar rats received a clip-compression SCI at C7. Within each of two study arms, animals were randomized into 5 groups (NPC, SAP, NPC+SAP, vehicle, and sham). SAPs and NPCs were injected into the spinal cord 1day and 14days post-injury, respectively. Animals received growth factors over 7days and were immunosuppressed. Rats were sacrificed at 4weeks and sections of the cervical spinal cord prepared for immunohistochemistry (first study arm). Neurological function was assessed weekly for 8weeks using a battery of behavioral tests. Nine weeks post-SCI, the corticospinal tract was assessed using fiber-tracking (second arm). RESULTS SAP-treated animals had significantly more surviving NPCs which showed increased differentiation to neurons and oligodendrocytes compared to controls. SAPs alone or in combination with NPCs resulted in smaller intramedullary cysts and larger volume of preserved tissue compared to other groups. The combined treatment group showed reduced astrogliosis and chondroitin sulfate proteoglycan deposition. Synaptic connectivity was increased in the NPC and combined treatment groups. Corticospinal tract preservation and behavioral outcomes improved with combinatorial treatment. CONCLUSION Injecting SAPs after SCI enhances subsequent NPC survival, integration and differentiation and improves functional recovery. STATEMENT OF SIGNIFICANCE The hostile environment after spinal cord injury (SCI) can compromise effects of regenerative therapies. We hypothesized that improving this environment with self-assembling peptides (SAPs) before neural precursor cell (NPC) transplantation would support their beneficial effects. SAPs assemble once injected, providing a supportive scaffold for repair and regeneration. We investigated this in a rat model of spinal cord injury. More NPCs survived in SAP-treated animals and these showed increased differentiation compared to controls. SAPS alone or in combination with NPCs resulted in smaller cysts and larger volume of preserved tissue with the combined treatment also reducing scarring and improving behavioral outcomes. Overall, injection of SAPs was shown to improve the efficacy of NPC treatment, a promising finding for those with SCIs.
The key signalling pathways and transcriptional programmes that instruct neuronal diversity during development have largely been identified. In this Review, we discuss how this knowledge has been used to successfully reprogramme various cell types into an amazing array of distinct types of functional neurons. We further discuss the extent to which direct neuronal reprogramming recapitulates embryonic development, and examine the particular barriers to reprogramming that may exist given a cell's unique developmental history. We conclude with a recently proposed model for cell specification called the 'Cook Islands' model, and consider whether it is a fitting model for cell specification based on recent results from the direct reprogramming field.
Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.
In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4–5 weeks.
Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.
Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.
Adult somatic stem cells in various organs maintain homeostatic tissue regeneration and enhance plasticity. Since its initial discovery five decades ago, investigations of adult neurogenesis and neural stem cells have led to an established and expanding field that has significantly influenced many facets of neuroscience, developmental biology, and regenerative medicine. Here we review recent progress and focus on questions related to adult mammalian neural stem cells that also apply to other somatic stem cells. We further discuss emerging topics that are guiding the field toward better understanding adult neural stem cells and ultimately applying these principles to improve human health.
Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.