Identification of human cells in brain xenografts and in neural co-cultures of rat by in situ hybridisation with Alu probe (original) (raw)
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Differentiated Neuronal Cell Lines as Donor Tissue for Transplantation into the CNS
Annals of the New York Academy of Sciences, 1987
We are investigating the use of specific neural cell lines for transplantation into the damaged central nervous system (CNS). Treatment of neuronal cell lines in vitro with various growth modulators and antimitotic drugs has been shown to render cells amitotic and to induce the normal neuronal phenotype.'-3 In this study, several mouse and human neuroblastomas and a rat pheochromocytoma were treated with either 0.5 pg/ml mitomycin C (mito) and lo-' M bromodeoxyuridine (Brdu) or 10 pg/ ml prostaglandin E I and 500 pg/ml dibutyryl cyclic adenosine monophosphate for examination of their neuronal properties before implantation into the lesioned CNS. The pheochromocytoma cells (PC12 cells) were first treated with 100 ng/ml nerve growth factor (NGF) to induce neuritic sprouting. Maximum neurite outgrowth occurred over 4 days, after which time PC12 cells were exposed to mito-Brdu for an additional 3 days. FIGURE la shows the normal appearance of mitotic PC12 cells. Cells are round with only a few anchoring extensions arising from some cells. When PC12 cells are treated as described, neuritic extensions can be quite long and branched. Significantly, removal of all drugs and NGF from the culture does not induce a reversion to the undifferentiated, mitotic state (FIG. Ib), and differentiated cells can be maintained in vitro for at least 1 month after drug removal. These cells show typical tyrosine hydroxylase immunocytochemical staining (FIG. lc) and produce 4.3 pg dopamine/mg protein as measured by high-performance liquid chromatography. N,AB-1 cells, a mouse neuroblastoma subclone of Neuro 2a, is an adrenergic line that extends bipolar, unbranched neurites for more than 50 pm when treated with either drug combination for 4 days. These cells become arrested in G,G, of the cell cycle as measured by flow cytometry of propidium iodide-stained DNA4 and show a significant change in cell surface glycoproteins. Living, control N,AB-1 cells bound fluorescein-labeled wheat germ agglutinin (FL-WGA) in a discontinuous pattern around the cell body (FIG. 2a) whereas differentiated cells showed an intense green fluorescence outlining both neurites and cell body completely (FIG. 2b). Differentiated aThis work was supported by Grants NS19711 (to M. F. D. Notter) and NS15109 (to D. M.
Methods, 2010
Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important to correlate "cell types" to gene expression. Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes. Importantly, we were able to combine ISH with retrograde tracing and antibody staining including BrdU staining that enables birthdating. These techniques should prove useful in identifying and characterizing the cell types of the neural tissues. In this article, we describe the methodology of these techniques, taking examples from our analyses of the mammalian cerebral cortex. 1. Introduction Central nervous system consists of a myriad of cell types, including neurons, glias, endothelial cells, etc. On top of it, each cell type can be further subdivided into many different subtypes [4,27]. Considering that the neuronal circuit is an assembly of various neuronal types, the identification and characterization of each subtype is central to the understanding of the circuit [13]. Recently, systematic efforts to map gene expression in the brain, such as Allen Brain Atlas ([22]; http://www.brain-map.org/), GENSAT ([11]; http://www.gensat.org/index.html) and others (e.g., genepaint.org; http://www.genepaint.org/Frameset.html) have revealed many candidate marker genes for cell type identification. Obviously, certain genes are specifically expressed by particular subsets of neurons. But what are the common features of these neurons? How are they related to the classical neuronal subtypes defined by morphology,
In Situ Hybridization in Human and Rodent Tissue by the Use of a New and Simplified Method
Applied Immunohistochemistry & Molecular Morphology, 2012
In situ hybridization (ISH) is a method that detects and localizes DNA or RNA in morphologically preserved tissue and cell preparations. The method is based on the principle that DNA or RNA will undergo hydrogen binding to complimentary sequences. Selective probes are labeled and used in order to detect specific sequences in tissues or cell preparations. Even though the method has improved over the past decades, there are still issues with sensitivity and specificity. The protocols are nonstandardized, and often time consuming due to multiple steps. In this paper, we have used a new and commercially available ISH kit for the detection of mRNA in formalin-fixed paraffin-embedded tissue. We have used both human and Mongolian gerbil tissue, and we evaluated mRNA expression of the neuroendocrine markers chromogranin A and histidine decarboxylase in both normal tissue and poorly differentiated tumor. In our experience, this method offers excellent sensitivity and specificity. The protocol is more standardized, and our results have been consistent. It is also less time consuming than conventional ISH protocols.
PLOS One, 2011
Background: Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).
In vitro culture and labeling of neural cell aggregates followed by transplantation
Experimental Neurology, 1987
Septal, cortical, or whole brain fetal (El 5-17) cells were dissociated and cultured in serum-supplemented Dulbecco minimum essential medium under rotating culture conditions. Preincubation and exposure to cytosine arabinoside was utilized to produce "neuron-rich" cultures. Fluorescent latex microbeads were added to cultures at seeding time or early after aggregate formation. All cell types were found to incorporate the fluorescent beads, although apparently not to the same extent. Two-to fiveday-old aggregates tended to attach and grow neurites after their transfer to poly-llysine-or Matrigel-coated dishes under stationary conditions. Early aggregates transplanted to the hippocampus of adult rats developed into identifiable grafts, with fluorescent-labeled cells. We conclude that "young" neural cell aggregates maintain their ability to undergo two basic phenomena for cellular interaction, i.e., attachment and neuritic growth. Floating aggregates may provide a convenient cellular condition whenever culturing of neural cells is to be used before grafting them into a host animal. 0 1987 Academic Press, Inc.
Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny
Scientific Reports, 2016
Many neurological injuries are likely too extensive for the limited repair capacity of endogenous neural stem cells (NSCs). An alternative is to isolate NSCs from a donor, and expand them in vitro as transplantation material. Numerous groups have already transplanted neural stem and precursor cells. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate in vivo. (2) There is little control over the cells' terminal differentiation, e.g., a graft intended to replace neurons might choose a predominantly glial fate. (3) There is limited ability of researchers to alter the combination of cell types in pursuit of a precise treatment. We demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in vitro, followed by isolation of the neuronal progeny. We transplanted undifferentiated hNPCs or a defined concentration of hNPC-derived neurons into mice, then compared these two groups with regard to their survival, proliferation and phenotypic fate. We present evidence suggesting that in vitro-differentiated-and-purified neurons survive as well in vivo as their undifferentiated progenitors, and undergo less proliferation and less astrocytic differentiation. We also describe techniques for optimizing low-temperature cell preservation and portability. Neurological diseases today afflict about a billion people, accounting for 12% of human mortalities worldwide, and the incidence is expected to rise with an aging population 1. The existence of stem cells in the adult mammalian 2 , particularly adult human 3 central nervous system (CNS) makes it feasible for neurological injuries to undergo repair by endogenous mechanisms. Unfortunately, adult neurogenesis is likely not robust enough to address the severity of many injuries 4,5. As another option, neural stem cells (NSCs) and precursor cells (NPCs) can be harvested from a donor, and then expanded in tissue culture for the purpose of later transplantation. Indeed, neural cell replacement therapy is a promising method to help regenerate the afflicted CNS, and the promise of this approach has inspired enormous amounts of global research. In light of the numerous types of neurodegenerative diseases and neurological insults diagnosed increasingly on an annual basis, it would seem that these research efforts are well placed. NSCs and NPCs have been transplanted as heterogeneous, undifferentiated material by many research groups, in animal models as well as clinically 1,4,6,7. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate deleteriously within the host 8,9. (2) There is little control over the donor cells' terminal differentiation, e.g., a graft intended to replace lost neurons might choose a predominantly glial fate 10-14. (3) There is insufficient ability of researchers to manage and modulate the specific combination of terminal cell types in pursuit of a precise injury treatment (i.e., there is limited investigative power). Controlling the terminal phenotypic fate of grafted cells has long been a challenge in the field. NSCs and NPCs implanted into the CNS have primarily become astrocytes 10-14 , which are inadequate by themselves to constitute neural networks and can even have adverse effects such as allodynia 10,15. Shortcomings such as these have inspired many groups to innovate ways of manipulating donor cells in vitro, prior to transplant, with the aim of enhancing transplant precision and functional outcome 16-18. Here we demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in tissue culture, followed by isolation of the neuronal progeny from the glia. We reasoned that by providing heterogeneous hNPCs with