Table 1 Milestones in the development of methods used to study proliferation and regeneration in mammalian tissues
From: Comparative regenerative mechanisms across different mammalian tissues
Start date | Strategies and tools | Purpose | Context of initial studies | Pros | Cons |
|---|---|---|---|---|---|
Equipment and technology | |||||
~1912 | Light microscopy | H&E1 staining and light microscopy used to measure the mitotic index of cells | Incidence of mitosis and cell division in the CNS2 and in lymphocytes | Specimen preparation is relatively short; can image live cells | Low resolution; specialized training required to identify mitotic phases; scoring cells is time-consuming |
~1970’s | Electron microscopy | Assess mitotic figures, cell morphology and tissue structure | Regeneration in intimal thickening, liver, adrenal cortex, and wound healing | High resolution | Specimen preparation and imaging requires specialized training; cannot use live cells; limited field of view |
~1970’s | Flow cytometry (FC) and fluorescence- activated cell sorting (FACS) | Rapidly assess cells expressing marker/s of interest and sort accordingly, e.g., cell cycle phase, or live/dead ratios | Sorting T and B lymphocyte populations; identifying cell cycle kinetics in lymphocytes | FC and FACS provide rapid data collection and quantification; increased statistical power with large cell numbers; FACs used to isolate cell populations of interest | Tissue disaggregation may result in cell losses and alter cell morphology; requires the use of well characterized markers; total loss-of-spatial information |
~1990’s | Laser scanning confocal microscopy | Spatiotemporal organization of cells in thick optical sections of specimens or isolated cells at high resolution | Cell configuration in the CNS2 to elucidate plasticity and function; organization of stress fibers in corneal wound healing | Three-dimensional visualization of specimen using tissue sections or isolated cells | Careful analysis of tissue sections is essential to correctly identify cell types and nuclei; requires the use of well characterized markers |
1995 | Microarrays | Determine transcripts expressed in tissues or cell populations | First publications of technique | High throughput and rapid analysis of relative expression levels of genes | Genes are selected a priori; the sensitivity range of gene expression levels is lower compared to RNA-Seq3 |
2007 and 2008 | RNA-Seq and ChIP-Seq4 | ChIP-Seq captures DNA bound to histone marks that affect gene expression; RNA-Seq is used to discover the transcriptome of cells or tissue | First publications of technique | Both RNA-Seq and ChIP-Seq are high throughput and large-scale techniques; RNA-Seq provides the most accurate and unbiased method to quantify gene expression | Generates a large amount of data that require bioinformatic expertize |
~2014 | High throughput single-cell RNA-Seq using microfluidics | Identify the heterogeneous characteristics of individual cells within a cell population in health and disease | Subsets of bone-marrow derived dendritic cells were found to modulate paracrine signaling with other cells | Determines genes expressed at a single-cell level; Drop-Seq5 is continually improving to become more accessible | Very expensive and time-consuming to construct many cDNA6 libraries (~100–1000 libraries); generates a large amount of data that require bioinformatic expertize |
Cell cycle kinetics | |||||
1935 | Colchicine | Arrests cells in metaphase of mitosis to measure the mitotic index | Characterization of tumor growth and the development of female reproductive organs | Greater chance of capturing cells in mitosis | Colchicine is toxic in high doses; non-specifically targets all cells; scoring mitotic cells is time-consuming |
~1960’s | Administration of tritiated ([3H])-thymidine | [3H]-thymidine is prospectively administered for incorporation during DNA synthesis (S-phase), indicative of cell cycle entry | Labeling DNA synthesis in mouse tissue (intestine, spleen, and pancreas); lineage tracing of osteoclast origins | [3H]-thymidine labeling principally identifies cells in S-phase and their [3H]-thymidine+ progeny | Specimen preparation and processing of autoradiographs is complex and time-consuming, and requires handling radioactive materials; does not signify cell proliferation in cells with incomplete progression through mitosis or cytokinesis; [3H]-thymidine labeling is dependent on the method of administration, dosage, and pulse-chase periods |
1984 | Antibody against Ki67, which is present in G1, S, G2, and M, but not in G0, indicating cell cycling | Marker of cell cycle activity | First publications of technique | Antibody can be used in multiple assays for the rapid assessment of cell cycle activity | Does not signify cell proliferation in cells with incomplete progression through mitosis or cytokinesis |
1982 | Antibody against BrdU7, which is a non-radioactive thymidine analog | BrdU is prospectively administered for incorporation during S-phase, indicative of cell cycle re-entry | First publications of technique | Antibody used in multiple assays for rapid assessment of cell cycle activity; BrdU principally identifies cells in S-phase and their BrdU+ progeny; does not require handling of radioactive material | Labels cells in S-phase; does not signify cell proliferation in cells with incomplete progression through mitosis or cytokinesis; BrdU signal is dependent on the method of administration, dosage, and pulse-chase period, which may affect the number of BrdU+ cells |
~2004 | Antibodies against phospho-histone 3 (pHH3) and Aurora B Kinase (AurkB) | Identifies cells in mitosis (pHH3) or cytokinesis (AurkB) | Hepatoblast proliferation in liver morphogenesis; grading cancer cell proliferation in meningiomas | Antibodies used in multiple assays for rapid assessment of cell cycle activity; definitively captures cells in mitosis (pHH3) or cytokinesis (AurkB is present in midbody region) | Labels cells in mitosis or cytokinesis (~80 min), capturing a fraction of cells cycling in this short period of time |
2005 | Birth-dating | Determine the birth date of cells by correlating 14C incorporation levels to atmospheric levels, which sharply increased with nuclear bomb testing and decays over time | Neurogenesis in the human brain | 14C is quantifiable in human tissue samples | Relies on mathematical algorithms to infer date, and hence, the resolution of cell birth date is ±2 human years |
Genetic toolbox | |||||
1985 | Genetically modified (GM) mice | The mouse genome is altered at the genomic sequence level affecting subsequent expression of the gene of interest | First publications of technique | GM mice represent a mammalian system that can be used to study gene effects in homeostasis and in response to injury and treatment; mice are cheaper to house compared to other mammals | Incomplete characterization of mouse model or inappropriate controls result in misleading conclusions; breeding programs are expensive and time-consuming |
1994–1999 | Conditional mutant mice (including the Cre-lox system) | Determine cell fate and/or the role of a gene in a spatiotemporal manner | First publications of technique | Allows tissue-specific or ubiquitous gene activation or knockout that is inducible with the simple administration of tamoxifen resulting in Cre recombinase translocation to the nucleus and recombination of genomic loxP sites | Expression of Cre may be ectopic or leaky leading to unwanted off-target effects; inefficient homologous recombination may lead to under-representation of the targeted cell population |
2007 | Brainbow/Confetti mice | Genetic lineage-tracing of specific cell types and their behavior i.e., clonogenicity | Fate mapping and spatiotemporal distribution of glial and neuronal cells; fate mapping of intestinal Lgr5+ stem cells during homeostatic self-renewal | The multiple configurations of fluorescent proteins yield over 90 spectral hues, allowing individual cells to be tagged under the same promoter | Spectral hue configurations are dependent on the transgene and the mouse breeding strategy; factors that alter spectral range, include the promoter fidelity, transgene copy no. and length, and the efficiency and duration of recombination |
2013 | CRISPR/Cas mediated genome editing in mammals | RNA-guided nuclease system used to rapidly generate GM mice | First publications of technique | Simple, efficient, affordable, and improved transgenesis with the rapid generation of GM mice compared to other genome editing technologies | Off-target mutations |
