Single cell biology beyond the era of antibodies: relevance, challenges, and promises in biomedical research

Abstract

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.

Potential applications of peptide nucleic acid in biomedical domain

“…Furthermore, the significance of single‐cell biology in basic research and translational medicine is growing at a faster pace; in which efficacious detection and isolation of specific subclasses using intracellular markers (IcM) are key for understanding their functional heterogeneity. The use of peptide nucleic acid probes (PNPs) as an IcMs against certain microRNA for cancer surgical margin predictions are gaining attention 16 . Though nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized such as xeno‐nucleic acids, threose nucleic acids, locked nucleic acids (LNA), morpholinos, and so on, along with PNA, 17‐21 among all these designed oligonucleotides, PNA is a remarkable nucleic acid mimic as it is neither a nucleic acids nor a peptide 22 .…”

“…The use of peptide nucleic acid probes (PNPs) as an IcMs against certain microRNA for cancer surgical margin predictions are gaining attention. 16 Though nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized such as xeno-nucleic acids, threose nucleic acids, locked nucleic acids (LNA), morpholinos, and so on, along with PNA, [17][18][19][20][21] among all these designed oligonucleotides, PNA is a remarkable nucleic acid mimic as it is neither a nucleic acids nor a peptide. 22 Furthermore, several reviews exist discussing PNA properties, 23 chemistry, 24,25 and its modifications [26][27][28] with details related to diagnostics and therapeutics, our review (Figure 2) specifically focuses on PNAs biomedical applications along with their properties, synthesis, and prospects.…”

The Use of Non-Apoptotic Sperm Selected by Magnetic Activated Cell Sorting (MACS) to Enhance Reproductive Outcomes: What the Evidence Says

“…Most of these separation approaches rely on fluorescent labelling, either using an antibody that specifically recognizes the target marker conjugated with a fluorescent agent or the expression of a genetically engineered fluorescent protein exclusively in the target cell type [27][28][29]. However, this approach is limited by the antibodies' availability, crossreactivity to other targets, and unspecific labelling [30][31][32]. Moreover, typically only surface proteins can be targeted, since antibodies and other recognition molecules are generally unable to cross the cell membrane.…”

Platforms for Single-Cell Collection and Analysis

“…The selection of cells of a certain type is often based on fluorescent labeling, either directly by a fluorescent antibody or by the expression of a protein genetically engineered to be fluorescent (green, yellow, or red) and specifically expressed in the targeted cell type [ 44 , 45 , 46 ]. The limitations of the first approach are available antibodies (particularly for less common organisms), cross-reactivity to other targets, and background unspecific labeling [ 47 , 48 , 49 ]. The second approach avoids many of these issues, but the identification of a suitable marker gene may be very complicated, especially under pathological conditions, when expression of cells is boosted and the presumptive marker can also appear in other cell types [ 50 , 51 , 52 ].…”