mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes

Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.     

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)^13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs^9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting¹⁶. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.     

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.     

Tracking and Predicting Human Somatic Cell Reprogramming Using Nuclear Characteristics

Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) generates valuable resources for disease modeling, toxicology, cell therapy, and regenerative medicine. However, the reprogramming process can be stochastic and inefficient, creating many partially reprogrammed intermediates and non-reprogrammed cells in addition to fully reprogrammed iPSCs. Much of the work to identify, evaluate, and enrich for iPSCs during reprogramming relies on methods that fix, destroy, or singularize cell cultures, thereby disrupting each cell’s microenvironment. Here, we develop a micropatterned substrate that allows for dynamic live-cell microscopy of hundreds of cell subpopulations undergoing reprogramming while preserving many of the biophysical and biochemical cues within the cells’ microenvironment. On this substrate, we were able to both watch and physically confine cells into discrete islands during the reprogramming of human somatic cells from skin biopsies and blood draws obtained from healthy donors. Using high-content analysis, we identified a combination of eight nuclear characteristics that can be used to generate a computational model to predict the progression of reprogramming and distinguish partially reprogrammed cells from those that are fully reprogrammed. This approach to track reprogramming in situ using micropatterned substrates could aid in biomanufacturing of therapeutically relevant iPSCs and be used to elucidate multiscale cellular changes (cell-cell interactions as well as subcellular changes) that accompany human cell fate transitions.     

免疫磁珠分选人角朊干细胞的实验研究

目的:探索人角朊干细胞(keratinocyte stem cells,KSCs)的免疫磁珠分选(Magnetic Activated Cell Sorting,MACS)的方法。方法:从人包皮组织获得角朊细胞悬液,然后利用人角朊干细胞表面CD49f(整合素α6)和CD71的表达情况(CD49fbriCD71dim)通过MACS法将KSCs分选出来,在荧光显微镜下观察其荧光标记情况,同时将分选所得的CD49fbriCD71dim细胞进行体外无血清培养,观察其形态及克隆形成。结果:人角朊细胞经MACS法分选后所得的CD49fbriCD71dim细胞比率可达12．02(±6．92)%。CD49fbriCD71dim细胞经培养可见细胞为典型的上皮样特征,呈铺路石样形态,高核浆比例,细胞紧密排列,轮廓清楚,折光性好,并可在5-7天左右形成全克隆,符合KSCs的特点。结论:研究表明MACS分选法可以得到较高比例的人角朊干细胞,并能进行后续培养研究,其不失为一种较理想的人角朊干细胞的分选方法。     

Expression of Retinoid X Receptors in Human Dermal Fibroblasts

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. In the observations presented here, we examined the regulation of expression of members of the RXR multigene family in human dermal fibroblasts. We showed that the messenger RNAs for both RXRα and RXRβ are expressed in human fibroblasts, but that the messenger RNA for RXRγ is not detectable in these cells. Electrophoretic mobility shift studies of binding to the β2RARE in human dermal fibroblasts demonstrated that a single complex binds to β2RARE in the absence of RA. Stimulating cells with all-transRA induced a second complex. An antibody to the RXRβ protein supershifted both complexes, while an antibody to the RXRα S/B protein had no effect on the binding. These data demonstrate that RXRβ plays an important role in retinoid-regulated signal transduction pathways in human dermal fibroblasts and the regulation of expression of the RXR gene family is different from that of the RAR gene family.     

Direct Generation of Neurosphere-Like Cells from Human Dermal Fibroblasts

Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted.     

Direct Reprogramming of Human Fibroblasts to Functional and Expandable Hepatocytes

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Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality.     

Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Ameliorates Ultraviolet A-Induced Damage of Normal Human Dermal Fibroblasts

Adult skin stem cells are considered an attractive cell resource for therapeutic potential in aged skin. We previously reported that multipotent human dermal stem/progenitor cells (hDSPCs) can be enriched from (normal human dermal fibroblasts (NHDFs) using collagen type IV. However, the beneficial effects of hDSPCs on aged skin remain to be elucidated. In the present study, we analyzed the growth factors secreted from hDSPCs in conditioned medium (CM) derived from hDSPCs (hDSPC-CM) and found that hDSPCs secreted higher levels of bFGF, IGFBP-1, IGFBP-2, HGF, VEGF and IGF-1 compared with non-hDSPCs. We then investigated whether hDSPC-CM has an effect on ultraviolet A (UVA)-irradiated NHDFs. Real-time RT-PCR analysis revealed that the treatment of UVA-irradiated NHDFs with hDSPC-CM significantly antagonized the UVA-induced up-regulation of the MMP1 and the UVA-induced down-regulation of the collagen types I, IV and V and TIMP1 mRNA expressions. Furthermore, a scratch wound healing assay showed that hDSPC-CM enhanced the migratory properties of UVA-irradiated NHDFs. hDSPC-CM also significantly reduced the number of the early and late apoptotic cell population in UVA-irradiated NHDFs. Taken together, these data suggest that hDSPC-CM can exert some beneficial effects on aged skin and may be used as a therapeutic agent to improve skin regeneration and wound healing.     

Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations.     

Fluorescently Activated Cell Sorting Followed by Microarray Profiling of Helper T Cell Subtypes from Human Peripheral Blood

Background

Peripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a) gene expression patterns in each cell type or (b) the proportion of cell types in blood. CD4⁺ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4⁺ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples.

Methodology/Principal Findings

We describe fluorescently activated cell sorting followed by microarray (FACS–array) technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers.

Conclusions/Significance

Our approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the involvement of a subpopulation of immune cells in some diseases.     

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected ^1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) ³. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.Download video file.^{(80M, mov)}