Methods to identify epithelial stem cells

Article information

Organoid. 2022;2.e24

Publication date (electronic) : 2022 September 25

doi : https://doi.org/10.51335/organoid.2022.2.e24

Youngtae Jeong

Department of New Biology and New Biology Research Center, DGIST, Daegu, Korea

Correspondence to: Youngtae Jeong, MD, PhD Department of New Biology, DGIST, E5-311, 333 Techno jungang-daero, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Korea E-mail: jyt@dgist.ac.kr

Received 2022 April 15; Revised 2022 August 16; Accepted 2022 August 29.

Abstract

Introduction

1. Epithelial tissue

The epithelium is a thin, continuous, compact layer of cells that line most of our body and organs’ inner and outer surfaces [1–4]. The epithelium protects the underlying tissues from mechanical insult, toxins, dryness, and radiation, and also provides sensation. The epithelium communicates with underlying tissues by secreting and exchanging various hormones and biochemicals. Depending on the number of layers, epithelial tissues are classified into simple epithelium, where every cell is in direct contact with the basement membrane in a single layer, and stratified epithelium, which is composed of multiple layers of epithelial cells. The shape of epithelial cells is defined as squamous (flat and more extended in width than in height), cuboidal (similar width and height, like a cube), or columnar (longer in height than in width). The combination of the cell shape and number of layers yields a classification of the following types of epithelium: simple squamous epithelium, simple cuboidal epithelium, simple columnar epithelium, stratified squamous epithelium, stratified cuboidal epithelium, stratified columnar epithelium, pseudostratified columnar epithelium, and transitional epithelium. Pseudostratified epithelium is essentially columnar epithelium in a single layer. However, it is called pseudostratified epithelium because the different height of nuclei confuses by making it seem like there are multiple layers of cells (where “pseudo” is a Greek prefix meaning “false”). Transitional epithelium has multiple layers of cells like stratified epithelium. However, the former has multiple shapes of cells, whereas the latter has one shape of cells. The types of epithelium, their characteristics, and their locations are summarized in Fig. 1.

Fig. 1.

Types of epithelium and their characteristics and locations.

One of the critical features of the epithelium is its fast turnover rate. In epithelial tissues such as the tongue, esophagus, and intestine, the turnover time takes only 3 to 7 days in a steady-state condition [5–7]. Even in some epithelial tissues with relatively slower turnover, regeneration is boosted upon injury. The maintenance of tissue integrity necessitates vigorous stem cell activity in the epithelium. The high turnover rate of the epithelium also leads to the development of cancers with much higher frequency than in connective, muscle, and nervous tissues. Furthermore, epithelial stem cells function as the cells of origin in many cancers [8–10]. Therefore, identifying epithelial stem cells and their self-renewal mechanisms is crucial for the maintenance of tissue integrity, the development of regenerative therapy, and the prevention of cancer.

2. Adult stem cells

Stem cells are undifferentiated cells that self-renew [11,12]. There are four major classes of stem cells that have these characteristics; embryonic stem cells, induced pluripotent stem cells, adult stem cells, and cancer stem cells. Because of their self-renewal capability, stem cells can reconstitute the tissues to which they originally belonged. Furthermore, the tissues or cells regenerated from a single stem cell have the same clonal characteristics.

Stem cells in the epithelium belong to the category of adult stem cells. In many epithelial tissues, cells facing outside are replenished from the bottom, implying that many epithelial stem cells reside close to the basement membrane. Therefore, experimental methods reflecting these characteristics have been developed and employed in order to identify tissue stem cells. The text below describes diverse methods used in stem cell identification, their rationales, and examples of their utility, which are summarized in Fig. 2.

Fig. 2.

Diverse methods to identify and study epithelial stem cells.

Label retaining

Previously, tissue stem cells had been thought to stay in a dormant state and start running the cell cycle only when necessary to maintain the tissue integrity or for repair in specific conditions such as injury. Therefore, label retention was considered a hallmark of tissue stem cells, and label-retaining cells (LRCs) were often interpreted as tissue stem cells [13,14]. To identify LRCs, BrdU or radioactive thymidine, which inserts in replicating DNA, was administered to mice for a certain period and traced for months or years to check whether any LRCs existed in the specific tissue. If there were no LRCs in a specific tissue, it was regarded that there existed no quiescent stem cells since the label was diluted by half upon every cell cycle. Thus, epithelial cells that retained the label longer than at least a month were considered stem cells in that tissue [15,16]. For example, LRCs were discovered in the basal layers of the palate, tongue, and skin, which were suggested as the location of keratinocyte stem cells [17,18]. Furthermore, subpopulations of cells including LRCs were more adherent and clonogenic [19–22]. In contrast, the lack of cells retaining labels over a month was interpreted as indicating the absence of stem cells in the top hierarchy [15]. However, the development of new stem cell research technologies, such as lineage tracing, made the stem cell identification process less dependent on label-retaining experiments. Furthermore, the identification of fast-cycling stem cells, such as LGR5+ intestinal stem cells, changed the concept of slow-cycling as a typical characteristic of tissue stem cells [23].

Asymmetric cell division

Stem cells are self-renewing cells, giving rise to one more-differentiated daughter cell and another cell maintaining the stem cell state. Although stem cell division can sometimes lead to the generation of two more-differentiated cells or two stem cells, the outcome is usually the generation of a stem cell and a more-differentiated cell [24]. This is often achieved by asymmetric cell division, which separates intracellular organelles and proteins unevenly to each cell. Therefore, epithelial stem cells were identified in the past by localizing cells with asymmetric cell division and rare mitosis. For example, esophageal stem cells were postulated to reside in the interpapillary basal layer (IBL) rather than in the papillary basal layer (PBL) since most cell divisions occur at right angles to the underlying basement membrane in the IBL, whereas cell divisions are symmetric in the PBL with a four times higher mitosis rate [25].

Clonogenic assay

Together with self-renewal, clonality is one of the main characteristics of stem cells. Thus, combined with a cell fractionation method such as fluorescence-activated cell sorting (FACS), clonogenic assays have been used to identify stem cell subpopulations. In particular, in human tissues where lineage tracing or transplantation experiments are restricted, clonogenic assays have served as one of the major assays for tissue stem cell identification.

Clonogenic assays include colony formation assays and organoids (Table 1). A colony is a group of more than 50 cells derived from one single cell [26,27]. Compared to the traditional two-dimensional cell line culture, colony formation assays have the advantage of reflecting clonality. Although they are 2-dimensional, 3-dimensional colonies can be generated using soft agar. Regardless of dimension, colony formation assays do not mimic their original histology. In contrast, organoids are stem cell-derived three-dimensional structures recapitulating the histology of their original tissues. Organoids include multiple types of cells in the tissue where the stem cells originate. They can be passaged multiple times, enabling them to be used to test stem cells’ self-renewal capability. Since prospective lineage tracing is not applicable to humans, organoid assays are among the most standard assays to identify and reflect human tissue stem cells. In detail, by applying different cell surface markers that divide epithelial cells into different subpopulations, epithelial cells can be FACS-sorted and cultured for organoid or colony formation. The subpopulation of cells that generate the highest efficiency of organoids or colonies is most enriched in epithelial stem cells. Indeed, esophageal, tracheal, and oral mucosal organoids were employed to identify their tissue stem cells [28–30].

Table 1.

Various in vitro 2D and 3D cell culture systems

However, several caveats exist in identifying stem cells based on clonogenic assays. First, progenitor cells, as well as stem cells, can also generate colonies or organoids. In the case of quiescent stem cells, early progenitors expand and begin to differentiate. Thus, early-stage progenitors can generate more organoids or colonies than dormant stem cells in some tissues. Second, the in vitro environment of clonogenic assays may not faithfully reflect the in vivo tissue environment and influences the generation of organoids or colonies. Therefore, although organoids reflect the critical aspects of stem cells, they should be combined with other experiments, such as gene expression and cell cycle analysis, when key in vivo experiments such as lineage tracing or transplantation are unavailable.

Lineage tracing

Lineage tracing is a technique to track all progeny of a single cell. Genetic lineage tracing involves labeling cells with reporters, usually expressing fluorescent proteins, activated by cell type-specific inducible Cre recombinase. Once labeled, all descendant cells of epithelial stem cells emit the same fluorescent signal, forming a fluorescent pillar and visualizing stem cells’ existence, location, and activity. Therefore, lineage tracing provides a powerful tool for identifying the tissue stem cells and their regulatory mechanisms.

A genetic lineage tracing system employs genome site-specific recombinases, usually a Cre/lox system [31]. Cycling recombinase (Cre ). Cre is a bacteriophage-derived recombinase that recognizes 34-base pair loxP sequences and induces excision, inversion, or translocation of sequences between loxP sites [32]. Cre-induced recombination is temporally regulated by fusing Cre with steroid hormone receptors such as estrogen receptor (ER) or progesterone receptor (PR). Since ER and PR are sequestered in the cytoplasm without their hormone ligands but translocate into the nucleus upon binding their own ligands, the CreER or CrePR fusion protein typically stays in the cytoplasm. If CreER or CrePR is inserted under a particular cell-specific promoter (CSP), CreER or CrePR is translated only in cells with vigorous CSP activity but still resides in the cytoplasm. Upon the binding of 4-hydroxytamoxifen (4-OHT) or mifepristone, CreER or CrePR can enter the nucleus, where Cre recombines genes between loxP sites. However, CreER or CrePR is not expressed in other cells without promoter activity.

Reporters are genetic constructs that express a marker gene upon translation. In the lineage tracing technology, genes encoding either fluorescent proteins such as green fluorescent protein (GFP) and tdTomato or chromogenic proteins such as LacZ are used. By placing the loxP-Stop codon-loxP (LSL) cassette in front of reporter genes and inserting it all together under the ROSA26 locus, we can ensure that transcription stops at the stop codon with reporter proteins untranscribed. However, when the LSL cassette is removed, the reporter proteins can be transcribed to label the cells. Although the initial fluorescent reporter system induced the new fluorescent signal, more advanced reporter systems were developed. mT/mG, double-fluorescent Cre reporter mice express a membrane-targeted tdTomato signal before Cre-mediated excision but express a membrane-targeted GFP after excision [33]. Confetti and Brainbow mice allow up to 4 and 9 different combinations of fluorescent signals through random recombination of tandem signals [34,35].

By mating mice with a CreER allele under the stem cell-specific promoter (sCSP) and mice with reporters under ROSA26, we can generate sCSP^CreER;R26^Reporter mice. In these mice, CreER is not transcribed in the differentiated cells since they do not have robust sCSP activity. In stem cells, however, CreER is expressed but still sequestered in the cytoplasm without tamoxifen injection. Thus, there is no reporter protein expression in any cells. However, upon tamoxifen injection, CreER in the cytoplasm binds to tamoxifen, moves into the nucleus, initiates recombination or deletion of the LSL cassette, and results in the reporter protein expression. Since epithelial stem cells self-renew and generate progeny, the size of clones of cells expressing the reporter protein increases over a certain amount of time.

The inducible conditional knockout system enables us to label and lineage-trace tissue stem cells and study the role of specific genes in tissue stem cells. After screening putative stem cell marker genes, we can generate CreER knock-in mice for these genes and cross them with reporter mice. By injecting tamoxifen and identifying which CreER mouse line provides the clonal pillar of a reporter gene that grows over time, we can identify the stem cells and their location in the tissue. For example, LGR5+ intestinal stem cells and Bmi1+ lingual stem cells were identified using LGR5-CreER and Bmi1-CreER mice [23,36]. Genetic signatures can also be identified by FACS of stem cells and differentiated cells and genomic or transcriptomic analyses. By crossing with conditional knockout mice of the gene of interest (GOI), the role of the GOI in the stem cells’ self-renewal or differentiation and their underlying mechanisms can be explored by comparing the pillars of reporter genes in the tissue, the transcriptomic profiles between the wild type and knockout of the GOI, and subsequent functional verification.

In addition to CreER or CrePR systems, there are other reporter systems. Flippase can also be bound to ER or PR and recombine FRT sequences [37]. In the Tet-on system, reverse tetracycline-controlled transcriptional activator can be expressed under the control of the CSP [38]. Dre recombinase recognizes the roxP sequence [39]. By combining two or more of these systems, the precision of genetic lineage tracing can be refined, and stem cells’ contribution to multiple tissues can be experimentally tested. For example, bronchioalveolar stem cells, which are located at the bronchioalveolar duct junction, were proposed to be the dual stem cells responsible for the lung alveoli and bronchioles [40]. Although clonal assays display self-renewal capability and multipotency [40,41], their direct contribution to both alveoli and bronchioles had not been experimentally demonstrated. After almost 15 years since their first discovery, the controversial existence and role of bronchioalveolar stem cells as dual stem cells for lung alveoli and bronchioles were finally proven by combining Cre/loxP and Dre/roxP systems [42].

In vivo tissue reconstitution after transplantation

Due to their self-renewal characteristics, stem cells have the tissue reconstitution capability in vivo. Therefore, transplantation of tissue stem cells with or without injury and confirmation of in vivo regeneration of the tissue to which the tissue stem cells originally belonged is a confirmatory experiment demonstrating the existence or enrichment of tissue stem cells. For example, transplantation of a single mammary stem cell into a mammary fat pad was employed to identify mammary gland regeneration by the mammary stem cell population [43,44]. However, injection of stem cells into their original organ is not technically possible in many organs (e.g., the trachea, intestine, and esophagus) with thin walls due to a high risk of puncture upon needle insertion. Therefore, many substitute transplantation methods have been developed and used, although the implantation sites are heterotopic.

Subrenal capsule transplantation is one of the most widely used methods to test tissue regeneration in stem cell biology. The kidney has a good blood flow that supports nutrition for the transplanted cells or tissues. In addition, the kidney capsule restricts the movement of transplanted cells or tissues and helps localize the transplantation site to verify tissue regeneration. Indeed, studies identifying several tissue stem cells, including esophageal and skeletal stem cells, employed this technique [29,45]. However, it requires surgical skills, which can be challenging to master in a short period. Furthermore, the renal capsule is also quite fragile, making handling technically more difficult.

Subcutaneous implantation is another method to test tissue regeneration after transplantation. It is technically more manageable than subrenal capsule transplantation. However, the blood flow in the subcutaneous tissue is not as abundant as in the kidney, so the successful implantation rate is lower than that of subrenal capsule transplantation. Furthermore, subcutaneous tissue allows a little movement of implanted cells or tissues. Therefore, finding the regenerated tissues in the implantation site, which is still less than millimeters in size, is very difficult. Some tissue sites with limited mobilization, such as the dorsal interscapular region, are preferred for this method [46].

There are additional tissue-specific methods to test transplantation and regeneration. As mentioned above, mammary stem cells are usually transplanted into mammary fat pads [43,44]. Intratracheal administration is used to study the regeneration of lung stem cells and progenitors [47,48]. In some studies, tracheal and esophageal stem cells are inoculated into devitalized rat tracheas, which are subsequently implanted under the dorsal skin of immunocompromised mice for weeks [49].

Gene expression pattern analysis

Measuring and comparing the expression of genes specifically expressed in stem cells or differentiated cells also provides important clues to identifying stem cells and their self-renewal mechanisms. Epithelial tissues comprise tissue stem cells and at least one or more differentiated cells. For example, the stratified squamous epithelium that lines the oral cavity, tongue, esophagus, skin, cornea, anus, and uterine cervix is composed of a basal layer, in or near which the immature stem cells reside, and suprabasal layers, in which cells undergo differentiation [29,36,50–52]. Several genes are differentially expressed between the basal layer and the suprabasal layers. In the stratified squamous epithelium, TP63, KRT14, KRT5, and SOX2 are exclusively expressed in the basal layer, and KRT1, KRT10, and LORICRIN are exclusively expressed in the suprabasal layer. The tissue stem cell subpopulation tends to have higher basal gene expression but lower suprabasal gene expression than other subpopulations. Furthermore, when the self-renewal mechanism is activated, basal gene expression is increased, but suprabasal gene expression is decreased. Therefore, measuring the expression of the marker genes for stem cells and differentiated cells can be employed to quickly assess the stem cell / differentiated cell subpopulations.

Cell surface markers

Lineage tracing is not applicable to the human body. Therefore, the identification of human tissue stem cells relies on identifying cell surface markers that allow the purification of stem cells at the single-cell level or at least the enrichment of stem cells. To this end, stem cell biologists conventionally applied different cell surface markers to dissociated epithelial cells. They used flow cytometry to analyze whether epithelial cells can be grouped into different subpopulations with antibodies for specific cell surface markers, and then sorted each subpopulation for direct functional experiments. Diverse methods, such as gene expression analysis, cell cycle analyses, clonogenic assay, and tissue reconstitution after transplantation, were employed to identify which subpopulation of cells is most enriched in stem cells.

The next issue is screening and identifying the potential surface markers for stem cells and differentiation cells. Before the advent of single-cell sequencing technology, potential cell surface markers were selected as below. In most epithelial tissues, the LRCs or asymmetric cells attach to the basement membrane. Therefore, components of laminins or hemidesmosomes such as α6 integrin (CD49f), β4 integrin (CD104), or other key cell adhesion molecules, including other integrins, were tested. Additionally, RNA sequencing (RNA-seq) of different subpopulations of cells was performed, and potential cell surface markers differentially expressed from each cell were selected and used for FACS. Then, the sorted groups of cells were tested for human stem cell identification. For example, RNA-seq was performed using Krt5-GFP positive and negative cells in mouse trachea, where Krt5 marks the basal cells [30]. Ngfr was selected from the RNA-seq analysis and confirmed as a murine tracheal stem cell marker after organoid assay and gene expression analysis. In the same paper, NGFR was also applied to human bronchial epithelial cells together with CD49f, and NGFR+CD49f+ cells formed human bronchial organoids with the highest efficiency. After identifying diverse tissue stem cells, it turned out that stem cells in different tissues can still have the same or similar cell surface marker profiles. Therefore, a pool of a handful of cell surface markers (NGFR, CD24, CD29, CD34, CD44, CD49f, CD71, CD104, CD133, EpCAM, Sca1, LGR5, LGR6, etc.) has been tested to identify cell surface markers for epithelial stem cells and differentiated cells [23,29,30,43,53–56].

However, recent technological advances have changed the identification process of stem cell surface markers. Single-cell RNA sequencing (scRNA-seq) enables us to easily divide and characterize epithelial cells into different subpopulations. Furthermore, we can easily check and compare the expression levels of the representative cell type-specific genes among different subpopulations, in order to identify stem cell and differentiated cell subpopulations. Then, we can use flow cytometry to analyze all epithelial cells with these markers, sort them, and perform functional assays. The combination of scRNA-seq and organoid systems will significantly advance the study of human epithelial stem cells.

Single-cell RNA sequencing

Cellular heterogeneity is an inherent feature of biological tissues since most tissues comprise tissue stem cells, cells under the differentiation processes, and terminally differentiated cells. Therefore, unraveling cellular heterogeneity is a prerequisite for tissue stem cell identification. scRNA-seq technology has greatly facilitated the process of cellular heterogeneity distinction. By applying previously cataloged stem cell- or differentiated cell-enriched gene sets, scRNA-seq further provides information on potential lineage hierarchy, regulatory mechanisms, and signaling networks between stem cells and stem cell niches [57,58]. Although it provides specialized information from a new angle, identifying stem cells using scRNA-seq still requires validation employing functional assays such as organoids or lineage tracing. In recent studies, the spatiotemporal scRNA-seq technique has been reported and applied to identify lineage differentiation of stem cells [59,60]. Once the resolution enhancement for the exact single-cell size is achieved, this technique will elucidate the detailed spatial information of epithelial stem cells and their differentiation process.

Conclusions

Epithelial stem cells are the major player responsible for epithelial tissue integrity maintenance and cancer development. The identification of epithelial stem cells and their self-renewal and differentiation mechanisms have long been of interest to stem cell biologists. Newly developed techniques have facilitated and advanced this process. However, many open questions still exist, in particular, regarding the identification and functional verification of human stem cells at the level of single-cell hierarchy and their exact tissue locations. Emerging techniques will unravel the detailed identity of stem cells from different perspectives and significantly advance our efforts to characterize epithelial stem cells.

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