RESUMO
Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Análise por Conglomerados , Simulação por Computador , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Software , Fluxo de TrabalhoRESUMO
In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 µm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.
Assuntos
Diferenciação Celular , Quitosana/química , Hidrogéis/química , Células-Tronco Neurais/citologia , Alicerces Teciduais/química , Acrilamidas/química , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/química , Fenômenos Químicos , Feminino , Proteínas Imobilizadas/química , Interferon gama/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/química , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/químicaRESUMO
Oligodendrocyte progenitor cell (OPC) culture has provided a powerful approach to mechanistically investigate the proliferation and differentiation of oligodendroglia. However, existing culture methods (including the traditional shake-off method) have limitations, particularly their low productivities. Therefore, we developed a simplified and highly efficient method to produce a large yield of OPCs with low expense by using specialised modified media, in which B104-conditioned medium (B104-CM) instead of growth factors was used as a mitogenic source for OPC propagation, while a modified OPC isolation-medium was applied to improve the isolation of OPCs. First, we withdrew foetal bovine serum when primary mixed glial cultures were 65-75% confluent and substituted with modified oligodendrocyte growth medium to enrich OPCs. Second, we employed a chemical-based method to isolate and purify OPCs from mixed glial cultures using a modified oligodendrocyte isolation medium. As a result, our approach produced a high yield of purified OPCs, approximately 90-fold higher than that produced via the traditional shake-off method. Importantly, the purified OPCs produced via our modified approach maintained typical capacities of proliferation and differentiation observed in oligodendrocyte lineage cells. Together, our modified method provides a highly efficient approach to OPC culture for oligodendrocyte research.
Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Oligodendroglia/citologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Western Blotting , Proliferação de Células , Imuno-Histoquímica , Microscopia Confocal , RatosRESUMO
Measuring the effects of sphingosine-1-phosphate (S1P) receptor modulators on human primary neural cells is of particular interest given the recent application of these central nervous system-accessible agents to the treatment of neurodegenerative diseases, such as multiple sclerosis. Issues to consider in experimental studies include the ability of some of these modulators to bind multiple S1P receptor subtypes simultaneously, the nonspecificity of commercially available S1P receptor antibodies, and activation of multiple intracellular signaling cascades by a given S1P receptor. Here, we discuss how to assay S1P receptor expression and activation using multiple agonists/antagonists, by linking the results of real-time reverse transcriptase polymerase chain reaction with the assessment of intracellular signaling derived from Western blot analyses.
Assuntos
Sistema Nervoso Central/citologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Western Blotting , Células Cultivadas , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Biotecnologia , Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/transplante , Oligodendroglia/citologia , Traumatismos da Medula Espinal/terapia , Transplante de Células-Tronco , Biotecnologia/economia , California , Linhagem Celular , Ensaios Clínicos como Assunto , Humanos , Neurogênese , Oligodendroglia/transplante , Recursos HumanosRESUMO
BACKGROUND: The purpose of this paper is two-fold. The first objective is to validate the assumptions behind a stochastic model developed earlier by these authors to describe oligodendrocyte generation in cell culture. The second is to generate time-lapse data that may help biomathematicians to build stochastic models of cell proliferation and differentiation under other experimental scenarios. RESULTS: Using time-lapse video recording it is possible to follow the individual evolutions of different cells within each clone. This experimental technique is very laborious and cannot replace model-based quantitative inference from clonal data. However, it is unrivalled in validating the structure of a stochastic model intended to describe cell proliferation and differentiation at the clonal level. In this paper, such data are reported and analyzed for oligodendrocyte precursor cells cultured in vitro. CONCLUSION: The results strongly support the validity of the most basic assumptions underpinning the previously proposed model of oligodendrocyte development in cell culture. However, there are some discrepancies; the most important is that the contribution of progenitor cell death to cell kinetics in this experimental system has been underestimated.